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Pierpont syndrome is a rare disorder characterized mainly by global developmental delay, unusual facial features, altered fat distribution in the limbs and hearing loss. A specific mutation (p.Tyr446Cys) in TBL1XR1, encoding a WD40 repeat-containing protein, which is a component of the SMRT/NCoR (silencing mediator retinoid and thyroid hormone receptors/nuclear receptor corepressors), has been reported as the genetic cause of Pierpont syndrome. Here, we used CRISPR-cas9 technology to generate a mutant mouse with the Y446C mutation in Tbl1xr1, which is also present in Pierpont syndrome. Several aspects of the phenotype were studied in the mutant mice: growth, body composition, hearing, motor behavior, thyroid hormone state and lipid and glucose metabolism. The mutant mice (Tbl1xr1Y446C/Y446C) displayed delayed growth, altered body composition with increased relative lean mass and impaired hearing. Expression of several genes involved in fatty acid metabolism differed in white adipose tissue, but not in liver or muscle of mutant mice compared to wild-type mice (Tbl1xr1+/+). No difference in thyroid hormone plasma concentrations was observed. Tbl1xr1Y446C/Y446C mice can be used as a model for distinct features of Pierpont syndrome, which will enable future studies on the pathogenic mechanisms underlying the various phenotypic characteristics.

BackgroundPatients with inactive thyroid hormone (TH) transporter MCT8 display intellectual disability due to an insufficient TH transport and action in the CNS. As a therapeutic strategy, application of Triac (3, 5, 3’-triiodothyroacetic acid) and Ditpa (3, 5 -diiodo-thyropropionic acid) have been proposed as both thyromimetic compounds are not dependent on MCT8 for cellular entry. Here, we tested and directly compared the thyromimetic actions of Triac versus Ditpa in Mct8/Oatp1c1 double knockout mice (Dko), a mouse model for human MCT8 deficiency.MethodsNewborn Dko mice were daily injected during the first three postnatal weeks with either Triac (50 ng/g or 400 ng/g) or Ditpa (400 ng/g or 4000 ng/g) and compared with Wt and Dko mice receiving saline injections. A second cohort of Dko mice was daily injected with Triac (400 ng/g) only between postnatal week 3 and 6. Thyromimetic effects in the CNS and peripheral tissues were monitored at different postnatal time points by immunofluorescence stainings for neural marker proteins, in situ hybridization and quantitative real time PCR. Locomotor performance was assessed in rotarod and hanging wire test. Acute brain slices of Triac treated Dko mice and their respective controls were used for electrophysiological recordings.ResultsOnly Dko mice injected with Triac (400 ng/g) during the first three postnatal weeks showed normalized myelination, differentiation of cortical GABAergic interneurons as well as locomotor performance. Electrophysiological recordings revealed an increased frequencies of cortical spontaneous miniature inhibitory postsynaptic currents in Dko mice and a normalization of this parameter in Triac treated Dko mice. In comparison, treatment of Dko mice with Ditpa at 4000 ng/g during the first three postnatal weeks resulted in normal myelination and cerebellar development but was less effective in restoring neuronal parameters and locomotor function. Finally, Triac was more potent than Ditpa in suppressingTrhandTshbexpression, respectively, and exerts stronger thyromimetic effects in liver and kidneys.ConclusionsIn newborn Dko deficient mice, Triac is highly effective and more efficient than Ditpa in promoting CNS maturation and function. Yet, Triac treatment needs to be initiated directly after birth to achieve the most beneficial effects.

Dysfunctions of the thyroid hormone (TH) transporting monocarboxylate transporter MCT8 lead to a complex X-linked syndrome with abnormal serum TH concentrations and prominent neuropsychiatric symptoms (Allan-Herndon-Dudley syndrome, AHDS). The key features of AHDS are replicated in double knockout mice lacking MCT8 and organic anion transporting protein OATP1C1 (Mct8/Oatp1c1 DKO). In this study, we characterize impairments of brain structure and function in Mct8/Oatp1c1 DKO mice using multimodal magnetic resonance imaging (MRI) and assess the potential of the TH analogue 3,3′,5-triiodothyroacetic acid (TRIAC) to rescue this phenotype. Structural and functional MRI were performed in 11-weeks-old male Mct8/Oatp1c1 DKO mice (N = 10), wild type controls (N = 7) and Mct8/Oatp1c1 DKO mice (N = 13) that were injected with TRIAC (400 ng/g bw s.c.) daily during the first three postnatal weeks. Grey and white matter volume were broadly reduced in Mct8/Oatp1c1 DKO mice. TRIAC treatment could significantly improve white matter thinning but did not affect grey matter loss. Network-based statistic showed a wide-spread increase of functional connectivity, while graph analysis revealed an impairment of small-worldness and whole-brain segregation in Mct8/Oatp1c1 DKO mice. Both functional deficits could be substantially ameliorated by TRIAC treatment. Our study demonstrates prominent structural and functional brain alterations in Mct8/Oatp1c1 DKO mice that may underlie the psychomotor deficiencies in AHDS. Additionally, we provide preclinical evidence that early-life TRIAC treatment improves white matter loss and brain network dysfunctions associated with TH transporter deficiency.

Thyroid hormone (TH) transporters are required for cellular transmembrane passage of TH and are thus mandatory for proper TH metabolism and action. Consequently, inactivating mutations in TH transporters such as MCT8 or OATP1C1 can cause tissue- specific changes in TH homeostasis. As the most prominent example, patients with MCT8 mutations exhibit elevated serum T3 levels, whereas their CNS appear to be in a TH deficient state. Here, we will briefly summarize recent studies of mice lacking Mct8 alone or in combination with the TH transporters Mct10 or Oatp1c1 that shed light on many aspects and pathogenic events underlying global MCT8 deficiency and also underscore the contribution of Mct10 and Oatp1c1 in tissue-specific TH transport processes. Moreover, development of conditional knock-out mice that allow a cell-specific inactivation of TH transporters in distinct tissues, disclosed cell-specific changes in TH signaling, thereby highlighting the pathophysiological significance of local control of TH action.

The function of the hippocampus depends on the process of adult hippocampal neurogenesis which underpins the exceptional neural plasticity of this structure, and is also frequently affected in CNS pathologies. Thus, manipulation of this process represents an important therapeutic goal. To identify potential strategies, organotypic adult brain slices are emerging as a valuable tool. Over the recent years, this methodology has been refined and here we present a combined protocol that brings together these refinements to enable long-term culture of adult hippocampal slices. We employ a sectioning technique that retains essential afferent inputs onto the hippocampus as well as serum-free culture conditions, so allowing an extended culture period. To sustain the neurogenic potential in the slices, we utilize the gliogenesis-inhibitor Indomethacin. Using EdU retention analysis enables us to assess the effects of pharmacological intervention on neurogenesis. With these improvements, we have established an easy and reliable method to study the effects of small molecules/drugs on proliferation and neuron formation ex vivo which will facilitate future discovery driven drug screenings.

OA Förderung 2022 Inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) result in Allan-Herndon-Dudley Syndrome, a severe form of psychomotor retardation, while inactivating mutations in another TH transporter, organic anion transporting polypeptide 1c1 (OATP1C1), are linked to juvenile neurodegeneration. These diseases point to essential roles for TH transporters in CNS function. We recently defined the presence of Mct8 in adult hippocampal progenitors and mature granule cell neurons and unraveled cell-autonomous and indirect requirements for Mct8 in adult hippocampal neurogenesis. Here, we investigated whether Oatp1c1 is involved in the hippocampal neurogenic process in concert with Mct8. We detected Oatp1c1 gene expression activity and transcripts in subsets of progenitors, neurons and niche cells in the dentate gyrus. Absence of Oatp1c1 resulted in increased neuroblast and reduced immature neuron numbers in 6-month-old Oatp1c1ko and Mct8/Oatp1c1 double knockout (M/Odko) mice. Reduced EdU-label retention in Mct8ko and M/Odko mice confirmed the impact of Mct8 on neuron formation. In contrast, no significant effect of Oatp1c1 loss on granule cell neuron production and anxiety-like behavior in the open field arena were seen. Together, our results reinforce that distinct actions of each TH transporter are required at multiple stages to ensure proper adult hippocampal neurogenesis.

The function of the hippocampus depends on the process of adult hippocampal neurogenesis which underpins the exceptional neural plasticity of this structure, and is also frequently affected in CNS pathologies. Thus, manipulation of this process represents an important therapeutic goal. To identify potential strategies, organotypic adult brain slices are emerging as a valuable tool. Over the recent years, this methodology has been refined and here we present a combined protocol that brings together these refinements to enable long-term culture of adult hippocampal slices. We employ a sectioning technique that retains essential afferent inputs onto the hippocampus as well as serum-free culture conditions, so allowing an extended culture period. To sustain the neurogenic potential in the slices, we utilize the gliogenesis-inhibitor Indomethacin. Using EdU retention analysis enables us to assess the effects of pharmacological intervention on neurogenesis. With these improvements, we have established an easy and reliable method to study the effects of small molecules/drugs on proliferation and neuron formation ex vivo which will facilitate future discovery driven drug screenings.

Summary Adult hippocampal neurogenesis is strongly dependent on thyroid hormone (TH). Whether TH signaling regulates this process in a cell-autonomous or non-autonomous manner remains unknown. To answer this question, we used global and conditional knockouts of the TH transporter monocarboxylate transporter 8 (MCT8), having first used FACS and immunohistochemistry to demonstrate that MCT8 is the only TH transporter expressed on neuroblasts and adult slice cultures to confirm a necessary role for MCT8 in neurogenesis. Both mice with a global deletion or an adult neural stem cell-specific deletion of MCT8 showed decreased expression of the cell-cycle inhibitor P27KIP1, reduced differentiation of neuroblasts, and impaired generation of new granule cell neurons, with global knockout mice also showing enhanced neuroblast proliferation. Together, our results reveal a cell-autonomous role for TH signaling in adult hippocampal neurogenesis alongside non-cell-autonomous effects on cell proliferation earlier in the lineage., Highlights • Thyroid hormone transporter MCT8 is expressed in the adult hippocampal lineage • MCT8 regulates neuroblast differentiation and neurogenesis cell-autonomously • Absence of MCT8 compromises expression of the cell-cycle regulator P27KIP1, In this article, Mayerl and colleagues show that the thyroid hormone transporter MCT8 is expressed by neuroblasts in the adult hippocampus where it acts as a gate-keeper for neuronal differentiation. By comparing the phenotypes of global and conditional knockout mice, they show that MCT8 acts cell-autonomously on neurogenesis via the regulation of the cell-cycle regulator P27KIP1, while having non-cell-autonomous effects on stem cells.

Although the underlying neurobiology of major mental illness (MMI) remains unknown, emerging evidence implicates a role for oligodendrocyte-myelin abnormalities. Here, we took advantage of a large family carrying a balanced t(1;11) translocation, which substantially increases risk of MMI, to undertake both diffusion tensor imaging (DTI) and cellular studies to evaluate the consequences of the t(1;11) translocation on white matter structural integrity and oligodendrocyte-myelin biology. This translocation disrupts among others theDISC1gene which plays a crucial role in brain development. We show that translocation-carrying patients display significant disruption in white matter integrity compared to familial controls. At a cellular level, we observe dysregulation of key pathways controlling oligodendrocyte development and morphogenesis in induced pluripotent stem cell (iPSC) case derived oligodendrocytes. This is associated with reduced proliferation and a stunted morphologyin vitro. Further, myelin internodes in a humanized mouse model that recapitulates the human translocation as well as after transplantation of t(1;11) oligodendrocyte progenitors were significantly reduced compared to controls. Thus we provide evidence that the t(1;11) translocation has biological effects at both the systems and cellular level that together suggest oligodendrocyte-myelin dysfunction.

Summary 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., Highlights • MCT8 and OATP1C1 expression increases upon activation of muscle stem cells • Loss of MCT8 and OATP1C1 expression inhibits muscle stem cell differentiation • Mct8- and Oatp1c1-deficient mice display impaired muscle regeneration, In this article, Mayerl and colleagues demonstrate that the thyroid hormone transporters MCT8 and OATP1C1 are unique gate-keepers in activated muscle stem cells. The expression of both transporters increases upon activation of muscle stem cells, while loss of MCT8 and OATP1C1 expression results in impaired muscle stem cell differentiation.

The monocarboxylate transporter Mct10 (Slc16a10; T-type amino acid transporter) facilitates the cellular transport of thyroid hormone (TH) and shows an overlapping expression with the well-established TH transporter Mct8. Because Mct8 deficiency is associated with distinct tissue-specific alterations in TH transport and metabolism, we speculated that Mct10 inactivation may compromise the tissue-specific TH homeostasis as well. However, analysis of Mct10 knockout (ko) mice revealed normal serum TH levels and tissue TH content in contrast to Mct8 ko mice that are characterized by high serum T3, low serum T4, decreased brain TH content, and increased tissue TH concentrations in the liver, kidneys, and thyroid gland. Surprisingly, mice deficient in both TH transporters (Mct10/Mct8 double knockout [dko] mice) showed normal serum T4 levels in the presence of elevated serum T3, indicating that the additional inactivation of Mct10 partially rescues the phenotype of Mct8 ko mice. As a consequence of the normal serum T4, brain T4 content and hypothalamic TRH expression were found to be normalized in the Mct10/Mct8 dko mice. In contrast, the hyperthyroid situation in liver, kidneys, and thyroid gland of Mct8 ko mice was even more severe in Mct10/Mct8 dko animals, suggesting that in these organs, both transporters contribute to the TH efflux. In summary, our data indicate that Mct10 indeed participates in tissue-specific TH transport and also contributes to the generation of the unusual serum TH profile characteristic for Mct8 deficiency.

The impact of thyroid hormone (TH) on metabolism and energy expenditure is well established, but the role of TH in regulating nutritional sensing, particularly in the central nervous system, is only poorly defined. Here, we studied the consequences of hypothyroidism on leptin production as well as leptin sensing in congenital hypothyroid TRH receptor 1 knockout (Trhr1 ko) mice and euthyroid control animals. Hypothyroid mice exhibited decreased circulating leptin levels due to a decrease in fat mass and reduced leptin expression in white adipose tissue. In neurons of the hypothalamic arcuate nucleus, hypothyroid mice showed increased leptin receptor Ob-R expression and decreased suppressor of cytokine signaling 3 transcript levels. In order to monitor putative changes in central leptin sensing, we generated hypothyroid and leptin-deficient animals by crossing hypothyroid Trhr1 ko mice with the leptin-deficient ob/ob mice. Hypothyroid Trhr1/ob double knockout mice showed a blunted response to leptin treatment with respect to body weight and food intake and exhibited a decreased activation of phospho-signal transducer and activator of transcription 3 as well as a up-regulation of suppressor of cytokine signaling 3 upon leptin treatment, particularly in the arcuate nucleus. These data indicate alterations in the intracellular processing of the leptin signal under hypothyroid conditions and thereby unravel a novel mode of action by which TH affects energy metabolism.

The monocarboxylate transporter 8 (MCT8) plays a critical role in mediating the uptake of thyroid hormones (THs) into the brain. In patients, inactivating mutations in the MCT8 gene are associated with a severe form of psychomotor retardation and abnormal serum TH levels. Here, we evaluate the therapeutic potential of the TH analog 3,5,3',5'-tetraiodothyroacetic acid (tetrac) as a replacement for T-4 in brain development. Using COS1 cells transfected with TH transporter and deiodinase constructs, we could show that tetrac, albeit not being transported by MCT8, can be metabolized to the TH receptor active compound 3,3',5-triiodothyroacetic acid (triac) by type 2 deiodinase and inactivated by type 3 deiodinase. Triac in turn is capable of replacing T-3 in primary murine cerebellar cultures where it potently stimulates Purkinje cell development. In vivo effects of tetrac were assessed in congenital hypothyroid Pax8-knockout (KO) and Mct8/Pax8 double-KO mice as well as in Mct8-KO and wild-type animals after daily injection of tetrac (400 ng/g body weight) during the first postnatal weeks. This treatment was sufficient to promote TH-dependent neuronal differentiation in the cerebellum, cerebral cortex, and striatum but was ineffective in suppressing hypothalamic TRH expression. In contrast, TSH transcript levels in the pituitary were strongly down-regulated in response to tetrac. Based on our findings we propose that tetrac administration offers the opportunity to provide neurons during the postnatal stage with a potent TH receptor agonist, thereby eventually reducing the neurological damage in patients with MCT8 mutations without deteriorating the thyrotoxic situation in peripheral tissues. (Endocrinology 154: 968-979, 2013)

The tumour suppressor Merlin, encoded by the gene NF2, is frequently mutated in the autosomal dominant disorder neurofibromatosis type II, characterised primarily by the development of schwannoma and other glial cell tumours. However, NF2 is expressed in virtually all analysed human and rodent organs, and its deletion in mice causes early embryonic lethality. Additionally, NF2 encodes for two major isoforms of Merlin of unknown functionality. Specifically, the tumour suppressor potential of isoform 2 remains controversial. In this study, we used Nf2 isoform-specific knockout mouse models to analyse the function of each isoform during development and organ homeostasis. We found that both isoforms carry full tumour suppressor functionality and can completely compensate the loss of the other isoform during development and in most adult organs. Surprisingly, we discovered that spermatogenesis is strictly dependent on the presence of both isoforms. While the testis primarily expresses isoform 1, we noticed an enrichment of isoform 2 in spermatogonial stem cells. Deletion of either isoform was found to cause decreased sperm quality as observed by maturation defects and head/midpiece abnormalities. These defects led to impaired sperm functionality as assessed by decreased sperm capacitation. Thus, we describe spermatogenesis as a new Nf2-dependent process. Additionally, we provide for the first time in vivo evidence for equal tumour suppressor potentials of Merlin isoform 1 and isoform 2.

Sulforhodamine 101 (SR101) is widely used for astrocyte identification, though the labeling mechanism remains unknown and the efficacy of labeling in different brain regions is heterogeneous. By combining region-specific isolation of astrocytes followed by transcriptome analysis, two-photon excitation microscopy, and mouse genetics, we identified the thyroid hormone transporter OATP1C1 as the SR101-uptake transporter in hippocampus and cortex. Electronic supplementary material The online version of this article (doi:10.1007/s00429-013-0645-0) contains supplementary material, which is available to authorized users.

Allan-Herndon-Dudley syndrome (AHDS), a severe form of psychomotor retardation with abnormal thyroid hormone (TH) parameters, is linked to mutations in the TH-specific monocarboxylate transporter MCT8. In mice, deletion of Mct8 (Mct8 KO) faithfully replicates AHDS-associated endocrine abnormalities; however, unlike patients, these animals do not exhibit neurological impairments. While transport of the active form of TH (T3) across the blood-brain barrier is strongly diminished in Mct8 KO animals, prohormone (T4) can still enter the brain, possibly due to the presence of T4-selective organic anion transporting polypeptide (OATP1C1). Here, we characterized mice deficient for both TH transporters, MCT8 and OATP1C1 (Mct8/Oatp1c1 DKO). Mct8/Oatp1c1 DKO mice exhibited alterations in peripheral TH homeostasis that were similar to those in Mct8 KO mice; however, uptake of both T3 and T4 into the brains of Mct8/Oatp1c1 DKO mice was strongly reduced. Evidence of TH deprivation in the CNS of Mct8/Oatp1c1 DKO mice included highly decreased brain TH content as well as altered deiodinase activities and TH target gene expression. Consistent with delayed cerebellar development and reduced myelination, Mct8/Oatp1c1 DKO mice displayed pronounced locomotor abnormalities. Intriguingly, differentiation of GABAergic interneurons in the cerebral cortex was highly compromised. Our findings underscore the importance of TH transporters for proper brain development and provide a basis to study the pathogenic mechanisms underlying AHDS. ispartof: Journal of Clinical Investigation vol:124 issue:5 pages:1987-1999 ispartof: location:United States status: published

Organic anion-transporting polypeptide 1c1 (Oatp1c1) (also known as Slco1c1 and Oatp14) belongs to the family of Oatp and has been shown to facilitate the transport of T-4. In the rodent brain, Oatp1c1 is highly enriched in capillary endothelial cells and choroid plexus structures where it may mediate the entry of T-4 into the central nervous system. Here, we describe the generation and first analysis of Oatp1c1-deficient mice. Oatp1c1 knockout (KO) mice were born with the expected frequency, were not growth retarded, and developed without any overt neurological abnormalities. Serum T-3 and T-4 concentrations as well as renal and hepatic deiodinase type 1 expression levels were indistinguishable between Oatp1c1 KO mice and control animals. Hypothalamic TRH and pituitary TSH mRNA levels were not affected, but brain T-4 and T-3 content was decreased in Oatp1c1-deficient animals. Moreover, increased type 2 and decreased type 3 deiodinase activities indicate a mild hypothyroid situation in the brain of Oatp1c1 KO mice. Consequently, mRNA expression levels of gene products positively regulated by T-3 in the brain were down-regulated. This central nervous system-specific hypothyroidism is presumably caused by an impaired passage of T-4 across the blood-brain barrier and indicates a unique function of Oatp1c1 in facilitating T-4 transport despite the presence of other thyroid hormone transporters such as Mct8. (Endocrinology 153: 1528-1537, 2012)