RESUMO
The key role of a thyroid hormone receptor in determining the maturation and diversity of cone photoreceptors reflects a profound influence of endocrine signaling on the cells that mediate color vision. However, the route by which hormone reaches cones remains enigmatic as cones reside in the retinal photoreceptor layer, shielded by the blood-retina barrier. Using genetic approaches, we report that cone differentiation is regulated by a membrane transporter for thyroid hormone, MCT8 (SLC16A2), in the retinal pigment epithelium (RPE), which forms the outer blood-retina barrier. Mct8-deficient mice display hypothyroid-like cone gene expression and compromised electroretinogram responses. Mammalian color vision is typically facilitated by cone types that detect medium-long (M) and short (S) wavelengths of light but Mct8-deficient mice have a partial shift of M to S cone identity, resembling the phenotype of thyroid hormone receptor deficiency. RPE-specific ablation of Mct8 results in similar shifts in cone identity and hypothyroid-like gene expression whereas reexpression of MCT8 in the RPE in Mct8-deficient mice partly restores M cone identity, consistent with paracrine-like control of thyroid hormone signaling by the RPE. Our findings suggest that in addition to transport of essential solutes and homeostatic support for photoreceptors, the RPE regulates the thyroid hormone signal that promotes cone-mediated vision.
Assuntos
Diferenciação Celular , Camundongos Knockout , Transportadores de Ácidos Monocarboxílicos , Células Fotorreceptoras Retinianas Cones , Epitélio Pigmentado da Retina , Simportadores , Animais , Células Fotorreceptoras Retinianas Cones/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Simportadores/metabolismo , Simportadores/genética , Epitélio Pigmentado da Retina/metabolismo , Camundongos , Hormônios Tireóideos/metabolismo , EletrorretinografiaRESUMO
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.
Assuntos
Proteínas Nucleares , Proteínas Repressoras , Animais , Deficiências do Desenvolvimento , Modelos Animais de Doenças , Fácies , Lipomatose , Camundongos , Mutação , Proteínas Nucleares/genética , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Proteínas Repressoras/genética , Hormônios TireóideosRESUMO
Proper CNS myelination depends on the timed availability of thyroid hormone (TH) that induces differentiation of oligodendrocyte precursor cells (OPCs) to mature, myelinating oligodendrocytes. Abnormal myelination is frequently observed in Allan-Herndon-Dudley syndrome caused by inactivating mutations in the TH transporter MCT8. Likewise, persistent hypomyelination is a key CNS feature of the Mct8/Oatp1c1 double knockout (Dko) mouse model, a well-established mouse model for human MCT8 deficiency that exhibits diminished TH transport across brain barriers and thus a TH deficient CNS. Here, we explored whether decreased myelin content is caused by an impairment in oligodendrocyte maturation. To that end, we studied OPC and oligodendrocyte populations in Dko mice versus wild-type and single TH transporter knockout animals at different developmental time points (at postnatal days P12, P30, and P120) using multi-marker immunostaining and confocal microscopy. Only in Dko mice we observed a reduction in cells expressing the oligodendroglia marker Olig2, encompassing all stages between OPCs and mature oligodendrocytes. Moreover, Dko mice exhibited at all analysed time points an increased portion of OPCs and a reduced number of mature oligodendrocytes both in white and grey matter regions indicating a differentiation blockage in the absence of Mct8/Oatp1c1. We also assessed cortical oligodendrocyte structural parameters by visualizing and counting the number of mature myelin sheaths formed per oligodendrocyte. Again, only Dko mice displayed a reduced number of myelin sheaths that in turn exhibited an increase in length indicating a compensatory response to the reduced number of mature oligodendrocytes. Altogether, our studies underscore an oligodendrocyte differentiation impairment and altered oligodendrocyte structural parameters in the global absence of Mct8 and Oatp1c1. Both mechanisms most likely do not only cause the abnormal myelination state but also contribute to compromised neuronal functionality in Mct8/Oatp1c1 deficient animals.
Assuntos
Simportadores , Animais , Humanos , Camundongos , Animais Geneticamente Modificados , Transportadores de Ácidos Monocarboxílicos/genética , Oligodendroglia , Simportadores/genética , Hormônios Tireóideos/genéticaRESUMO
A genetic deficiency of the solute carrier monocarboxylate transporter 8 (MCT8), termed Allan-Herndon-Dudley syndrome, is an important cause of X-linked intellectual and motor disability. MCT8 transports thyroid hormones across cell membranes. While thyroid hormone analogues improve peripheral changes of MCT8 deficiency, no treatment of the neurological symptoms is available so far. Therefore, we tested a gene replacement therapy in Mct8- and Oatp1c1-deficient mice as a well-established model of the disease. Here, we report that targeting brain endothelial cells for Mct8 expression by intravenously injecting the vector AAV-BR1-Mct8 increased tri-iodothyronine (T3) levels in the brain and ameliorated morphological and functional parameters associated with the disease. Importantly, the therapy resulted in a long-lasting improvement in motor coordination. Thus, the data support the concept that MCT8 mediates the transport of thyroid hormones into the brain and indicate that a readily accessible vascular target can help overcome the consequences of the severe disability associated with MCT8 deficiency.
Assuntos
Pessoas com Deficiência , Deficiência Intelectual Ligada ao Cromossomo X , Transtornos Motores , Simportadores , Camundongos , Animais , Humanos , Barreira Hematoencefálica/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Hipotonia Muscular/genética , Atrofia Muscular , Células Endoteliais/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Hormônios Tireóideos/metabolismo , Terapia Genética , Simportadores/genética , Simportadores/metabolismoRESUMO
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.
Assuntos
Transportadores de Ácidos Monocarboxílicos , Simportadores , Animais , Proteínas Hedgehog/metabolismo , Hibridização in Situ Fluorescente , Interneurônios/metabolismo , Camundongos , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/genética , Simportadores/metabolismo , Hormônios Tireóideos/metabolismoRESUMO
Patients with inactive thyroid hormone (TH) transporter MCT8 display intellectual disability due to compromised central TH transport and action. As a therapeutic strategy, application of thyromimetic, MCT8-independent compounds Triac (3,5,3'-triiodothyroacetic acid), and Ditpa (3,5-diiodo-thyropropionic acid) was proposed. Here, we directly compared their thyromimetic potential in Mct8/Oatp1c1 double knock-out mice (Dko) modeling human MCT8 deficiency. Dko mice received either Triac (50 ng/g or 400 ng/g) or Ditpa (400 ng/g or 4000 ng/g) daily during the first three postnatal weeks. Saline-injected Wt and Dko mice served as controls. A second cohort of Dko mice received Triac (400 ng/g) daily between postnatal weeks 3 and 6. Thyromimetic effects were assessed at different postnatal stages by immunofluorescence, ISH, qPCR, electrophysiological recordings, and behavior tests. Triac treatment (400 ng/g) induced normalized myelination, cortical GABAergic interneuron differentiation, electrophysiological parameters, and locomotor performance only when administered during the first three postnatal weeks. Ditpa (4000 ng/g) application to Dko mice during the first three postnatal weeks resulted in normal myelination and cerebellar development but only mildly improved neuronal parameters and locomotor function. Together, Triac is highly-effective and more efficient than Ditpa in promoting CNS maturation and function in Dko mice yet needs to be initiated directly after birth for the most beneficial effects.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X , Simportadores , Animais , Camundongos , Deficiência Intelectual Ligada ao Cromossomo X/tratamento farmacológico , Camundongos Knockout , Transportadores de Ácidos Monocarboxílicos , Neurogênese , Hormônios Tireóideos/uso terapêuticoRESUMO
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.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X , Simportadores , Substância Branca , Animais , Masculino , Camundongos , Substância Branca/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Hormônios Tireóideos/metabolismo , Atrofia Muscular/metabolismo , Camundongos Knockout , Deficiência Intelectual Ligada ao Cromossomo X/tratamento farmacológico , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMO
Inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) causes a rare and debilitating form of X-linked psychomotor disability known as Allan Herndon Dudley syndrome (AHDS). One of the most prominent pathophysiological symptoms of MCT8-deficiency is hypomyelination. Here, patient-derived induced pluripotent stem cells (iPSCs) were used to study the role of MCT8 and TH on the maturation of oligodendrocytes. Interestingly, neither MCT8 mutations nor reduced TH affected the in vitro differentiation of control or MCT8-deficient iPSCs into oligodendrocytes. To assess whether patient-derived iPSC-derived oligodendrocyte progenitor cells (iOPCs) could provide myelinating oligodendrocytes in vivo, cells were transplanted into the shiverer mouse corpus callosum where they survived, migrated, and matured into myelinating oligodendrocytes, though the myelination efficiency was reduced compared with control cells. When MCT8-deficient and healthy control iOPCs were transplanted into a novel hypothyroid immunodeficient triple knockout mouse (tKO, mct8-/- ; oatp1c1-/- ; rag2-/- ), they failed to provide behavioral recovery and did not mature into oligodendrocytes in the hypothyroid corpus callosum, demonstrating the critical role of TH transport across brain barriers in oligodendrocyte maturation. We conclude that MCT8 plays a cell autonomous role in oligodendrocyte maturation and that functional TH transport into the central nervous system will be required for developing an effective treatment for MCT8-deficient patients.
Assuntos
Células Precursoras de Oligodendrócitos , Simportadores , Animais , Encéfalo/metabolismo , Membrana Celular/metabolismo , Humanos , Camundongos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Simportadores/genética , Simportadores/metabolismo , Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismoRESUMO
Cathepsin K-mediated thyroglobulin proteolysis contributes to thyroid hormone (TH) liberation, while TH transporters like Mct8 and Mct10 ensure TH release from thyroid follicles into the blood circulation. Thus, thyroid stimulating hormone (TSH) released upon TH demand binds to TSH receptors of thyrocytes, where it triggers Gαq-mediated short-term effects like cathepsin-mediated thyroglobulin utilization, and Gαs-mediated long-term signaling responses like thyroglobulin biosynthesis and thyrocyte proliferation. As reported recently, mice lacking Mct8 and Mct10 on a cathepsin K-deficient background exhibit excessive thyroglobulin proteolysis hinting towards altered TSH receptor signaling. Indeed, a combination of canonical basolateral and non-canonical vesicular TSH receptor localization was observed in Ctsk-/-/Mct8-/y/Mct10-/- mice, which implies prolonged Gαs-mediated signaling since endo-lysosomal down-regulation of the TSH receptor was not detected. Inspection of single knockout genotypes revealed that the TSH receptor localizes basolaterally in Ctsk-/- and Mct8-/y mice, whereas its localization is restricted to vesicles in Mct10-/- thyrocytes. The additional lack of cathepsin K reverses this effect, because Ctsk-/-/Mct10-/- mice display TSH receptors basolaterally, thereby indicating that cathepsin K and Mct10 contribute to TSH receptor homeostasis by maintaining its canonical localization in thyrocytes. Moreover, Mct10-/- mice displayed reduced numbers of dead thyrocytes, while their thyroid gland morphology was comparable to wild-type controls. In contrast, Mct8-/y, Mct8-/y/Mct10-/-, and Ctsk-/-/Mct8-/y/Mct10-/- mice showed enlarged thyroid follicles and increased cell death, indicating that Mct8 deficiency results in altered thyroid morphology. We conclude that vesicular TSH receptor localization does not result in different thyroid tissue architecture; however, Mct10 deficiency possibly modulates TSH receptor signaling for regulating thyrocyte survival.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Receptores da Tireotropina/metabolismo , Células Epiteliais da Tireoide/metabolismo , Glândula Tireoide/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/deficiência , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Catepsina K/deficiência , Catepsina K/genética , Catepsina K/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Tireoglobulina/metabolismo , Glândula Tireoide/citologia , Hormônios Tireóideos/metabolismo , Tireotropina/sangue , Tireotropina/metabolismoRESUMO
The thyroid gland is both a thyroid hormone (TH) generating as well as a TH responsive organ. It is hence crucial that cathepsin-mediated proteolytic cleavage of the precursor thyroglobulin is regulated and integrated with the subsequent export of TH into the blood circulation, which is enabled by TH transporters such as monocarboxylate transporters Mct8 and Mct10. Previously, we showed that cathepsin K-deficient mice exhibit the phenomenon of functional compensation through cathepsin L upregulation, which is independent of the canonical hypothalamus-pituitary-thyroid axis, thus, due to auto-regulation. Since these animals also feature enhanced Mct8 expression, we aimed to understand if TH transporters are part of the thyroid auto-regulatory mechanisms. Therefore, we analyzed phenotypic differences in thyroid function arising from combined cathepsin K and TH transporter deficiencies, i.e., in Ctsk-/-/Mct10-/-, Ctsk-/-/Mct8-/y, and Ctsk-/-/Mct8-/y/Mct10-/-. Despite the impaired TH export, thyroglobulin degradation was enhanced in the mice lacking Mct8, particularly in the triple-deficient genotype, due to increased cathepsin amounts and enhanced cysteine peptidase activities, leading to ongoing thyroglobulin proteolysis for TH liberation, eventually causing self-thyrotoxic thyroid states. The increased cathepsin amounts were a consequence of autophagy-mediated lysosomal biogenesis that is possibly triggered due to the stress accompanying intrathyroidal TH accumulation, in particular in the Ctsk-/-/Mct8-/y/Mct10-/- animals. Collectively, our data points to the notion that the absence of cathepsin K and Mct8 leads to excessive thyroglobulin degradation and TH liberation in a non-classical pathway of thyroid auto-regulation.
Assuntos
Autofagia/fisiologia , Catepsina K/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/metabolismo , Tireoglobulina/metabolismo , Glândula Tireoide/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Transporte Biológico , Catepsina L/metabolismo , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hipófise/metabolismoRESUMO
Neocortex expansion during evolution is associated with the enlargement of the embryonic subventricular zone, which reflects an increased self-renewal and proliferation of basal progenitors. In contrast to human, the vast majority of mouse basal progenitors lack self-renewal capacity, possibly due to lack of a basal process contacting the basal lamina and downregulation of cell-autonomous production of extracellular matrix (ECM) constituents. Here we show that targeted activation of the ECM receptor integrin αvß3 on basal progenitors in embryonic mouse neocortex promotes their expansion. Specifically, integrin αvß3 activation causes an increased cell cycle re-entry of Pax6-negative, Tbr2-positive intermediate progenitors, rather than basal radial glia, and a decrease in the proportion of intermediate progenitors committed to neurogenic division. Interestingly, integrin αvß3 is the only known cell surface receptor for thyroid hormones. Remarkably, tetrac, a thyroid hormone analog that inhibits the binding of thyroid hormones to integrin αvß3, completely abolishes the intermediate progenitor expansion observed upon targeted integrin αvß3 activation, indicating that this expansion requires the binding of thyroid hormones to integrin αvß3. Convergence of ECM and thyroid hormones on integrin αvß3 thus appears to be crucial for cortical progenitor proliferation and self-renewal, and hence for normal brain development and the evolutionary expansion of the neocortex.
Assuntos
Integrina alfaVbeta3/metabolismo , Neocórtex/embriologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Hormônios Tireóideos/metabolismo , Animais , Matriz Extracelular/metabolismo , Citometria de Fluxo , Fluorescência , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/metabolismo , Células-Tronco Neurais/metabolismo , Compostos de Fenilureia , Ratos , Ratos Sprague-Dawley , Proteínas com Domínio T/metabolismo , Tiroxina/análogos & derivadosRESUMO
Food supplements based on herbal products are widely used during pregnancy as part of a self-care approach. The idea that such supplements are safe and healthy is deeply seated in the general population, although they do not underlie the same strict safety regulations than medical drugs. We aimed to characterize the neurodevelopmental effects of the green tea catechin epigallocatechin gallate (EGCG), which is now commercialized as high-dose food supplement. We used the "Neurosphere Assay" to study the effects and unravel underlying molecular mechanisms of EGCG treatment on human and rat neural progenitor cells (NPCs) development in vitro. EGCG alters human and rat NPC development in vitro. It disturbs migration distance, migration pattern, and nuclear density of NPCs growing as neurospheres. These functional impairments are initiated by EGCG binding to the extracellular matrix glycoprotein laminin, preventing its binding to ß1-integrin subunits, thereby prohibiting cell adhesion and resulting in altered glia alignment and decreased number of migrating young neurons. Our data raise a concern on the intake of high-dose EGCG food supplements during pregnancy and highlight the need of an in vivo characterization of the effects of high-dose EGCG exposure during neurodevelopment.
Assuntos
Catequina/análogos & derivados , Células-Tronco Neurais/efeitos dos fármacos , Animais , Catequina/administração & dosagem , Catequina/efeitos adversos , Catequina/metabolismo , Catequina/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Suplementos Nutricionais , Feminino , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Integrina beta1/metabolismo , Laminina/metabolismo , Nestina/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Gravidez , RatosRESUMO
BACKGROUND: As a prerequisite for thyroid hormone (TH) metabolism and action TH has to be transported into cells where TH deiodinases and receptors are located. The trans-membrane passage of TH is facilitated by TH transporters of which the monocarboxylate transporter MCT8 has been most intensively studied. Inactivating mutations in the gene encoding MCT8 are associated with a severe form of psychomotor retardation and abnormal serum TH levels (Allan-Herndon-Dudley syndrome). In order to define the underlying pathogenic mechanisms, Mct8 knockout mice have been generated and intensively studied. Most surprisingly, Mct8 ko mice do not show any neurological symptoms but fully replicate the abnormal serum thyroid state. SCOPE OF REVIEW: We will summarize the findings of these mouse studies that shed light on various aspects of Mct8 deficiency and unambiguously demonstrated the pivotal role of Mct8 in mediating TH transport in various tissues. These studies have also revealed the presence of the complex interplay between different pathogenic mechanisms that contribute to the generation of the abnormal TH serum profile. MAJOR CONCLUSIONS: Most importantly, studies of Mct8 ko mice indicated the presence of additional TH transporters that act in concert with Mct8. Interesting candidates for such a function are the L-type amino acid transporters Lat1 and Lat2 as well as the organic anion transporting polypeptide Oatp1c1. GENERAL SIGNIFICANCE: Overall, the analysis of Mct8 deficient mice has greatly expanded our knowledge about the (patho-) physiological function of this transporter and established a sound basis for the characterization of additional TH transporter candidates. This article is part of a Special Issue entitled Thyroid hormone signalling.
Assuntos
Proteínas de Membrana Transportadoras/deficiência , Hormônios Tireóideos/metabolismo , Animais , Transporte Biológico , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Transportadores de Ácidos Monocarboxílicos/deficiência , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores , Hormônios Tireóideos/genéticaRESUMO
Introduction: Thyroid hormone transporters are essential for thyroid hormones to enter target cells. Monocarboxylate transporter (MCT) 8 is a key transporter and is expressed at the blood-brain barrier (BBB), in neural cells and many other tissues. Patients with MCT8 deficiency have severe neurodevelopmental delays because of cerebral hypothyroidism and chronic sequelae of peripheral thyrotoxicosis. The T3 analog 3,3',5-triiodothyroacetic acid (TRIAC) rescued neurodevelopmental features in animal models mimicking MCT8 deficiency and improved key metabolic features in patients with MCT8 deficiency. However, the identity of the transporter(s) that facilitate TRIAC transport are unknown. Here, we screened candidate transporters that are expressed at the human BBB and/or brain-cerebrospinal fluid barrier and known thyroid hormone transporters for TRIAC transport. Materials and Methods: Plasma membrane expression was determined by cell surface biotinylation assays. Intracellular accumulation of 1 nM TRIAC was assessed in COS-1 cells expressing candidate transporters in Dulbecco's phosphate-buffered saline (DPBS)/0.1% glucose or Dulbecco's modified Eagle's medium (DMEM) with or without 0.1% bovine serum albumin (BSA). Expression of Slc22a8 was determined by fluorescent in situ hybridization in brain sections from wild-type and Mct8/Oatp1c1 knockout mice at postnatal days 12, 21, and 120. Results: In total, 59 plasma membrane transporters were selected for screening of TRIAC accumulation (n = 40 based on expression at the human BBB and/or brain-cerebrospinal fluid barrier and having small organic molecules as substrates; n = 19 known thyroid hormone transporters). Screening of the selected transporter panel showed that 18 transporters facilitated significant intracellular accumulation of TRIAC in DPBS/0.1% glucose or DMEM in the absence of BSA. In the presence of BSA, substantial transport was noted for SLCO1B1 and SLC22A8 (in DPBS/0.1% glucose and DMEM) and SLC10A1, SLC22A6, and SLC22A24 (in DMEM). The zebrafish and mouse orthologs of these transporters similarly facilitated intracellular accumulation of TRIAC. Highest Slc22a8 mRNA expression was detected in mouse brain capillary endothelial cells and choroid plexus epithelial cells at early postnatal time points, but was reduced at P120. Conclusions: Human SLC10A1, SLCO1B1, SLC22A6, SLC22A8, and SLC22A24 as well as their mouse and zebrafish orthologs are efficient TRIAC transporters. These findings contribute to the understanding of TRIAC treatment in patients with MCT8 deficiency and animal models thereof.
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The impact of special histological types (ST) in triple-negative breast cancer (TNBC) and its association with overall outcome has gained increasing relevance as survival has been linked to specific histological TNBC subtypes. We evaluated the clinicopathological and survival data of 598 patients with 613 TNBCs, including 464 TNBCs of no special type (NST) and 149 TNBCs ST (low-grade, n = 12, 8.1%; high-grade, n = 112, 75.2%; apocrine and androgen receptor-positive [APO AR], n = 25, 16.8%). Patients with low-grade TNBC ST and TNBC ST APO AR were significantly older (P < 0.001) and had a lower Ki67 index (P < 0.001) than those with TNBC NST. Patients with high-grade TNBC ST were significantly older (P = 0.006) and had poorer pathological responses to neoadjuvant chemotherapy (NAC) (P < 0.001) than those with TNBC NST. Significant survival differences were observed between low-grade TNBC ST, TNBC ST APO AR, high-grade TNBC ST, and TNBC NST in the entire study group (DFS, P = 0.002; DDFS, P = 0.001) and in the non-NAC subgroup (OS, P = 0.034; DFS, P = 0.001; DDFS, P < 0.001). Patients with low-grade TNBC ST had the best survival outcomes. Patients with high-grade TNBC ST showed significantly worse outcomes than those with TNBC NST (entire study group: OS, P = 0.049; DFS, P < 0.001; DDFS, P = 0.001; non-NAC subgroup: OS, P = 0.014; DFS, P < 0.001; DDFS, P < 0.001). We conclude that prognostic stratification of TNBC ST is ultimately important for optimizing the therapeutic management of patients with these rare tumor entities.
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PURPOSE: The OlympiA randomized phase III trial compared 1 year of olaparib (OL) or placebo (PL) as adjuvant therapy in patients with germline BRCA1/2, high-risk human epidermal growth factor receptor 2-negative early breast cancer after completing (neo)adjuvant chemotherapy ([N]ACT), surgery, and radiotherapy. The patient-reported outcome primary hypothesis was that OL-treated patients may experience greater fatigue during treatment. METHODS: Data were collected before random assignment, and at 6, 12, 18, and 24 months. The primary end point was fatigue, measured with the Functional Assessment of Chronic Illness Therapy-Fatigue scale. Secondary end points, assessed with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire, Core 30 item, included nausea and vomiting (NV), diarrhea, and multiple functional domains. Scores were compared between treatment groups using mixed model for repeated measures. Two-sided P values <.05 were statistically significant for the primary end point. All secondary end points were descriptive. RESULTS: One thousand five hundred and thirty-eight patients (NACT: 746, ACT: 792) contributed to the analysis. Fatigue severity was statistically significantly greater for OL versus PL, but not clinically meaningfully different by prespecified criteria (≥3 points) at 6 months (diff OL v PL: NACT: -1.3 [95% CI, -2.4 to -0.2]; P = .022; ACT: -1.3 [95% CI, -2.3 to -0.2]; P = .017) and 12 months (NACT: -1.6 [95% CI, -2.8 to -0.3]; P = .017; ACT: -1.3 [95% CI, -2.4 to -0.2]; P = .025). There were no significant differences in fatigue severity between treatment groups at 18 and 24 months. NV severity was worse in patients treated with OL compared with PL at 6 months (NACT: 6.0 [95% CI, 4.1 to 8.0]; ACT: 5.3 [95% CI, 3.4 to 7.2]) and 12 months (NACT: 6.4 [95% CI, 4.4 to 8.3]; ACT: 4.5 [95% CI, 2.8 to 6.1]). During treatment, there were some clinically meaningful differences between groups for other symptoms but not for function subscales or global health status. CONCLUSION: Treatment-emergent symptoms from OL were limited, generally resolving after treatment ended. OL- and PL-treated patients had similar functional scores, slowly improving during the 24 months after (N)ACT and there was no clinically meaningful persistence of fatigue severity in OL-treated patients.
Assuntos
Neoplasias da Mama , Ftalazinas , Piperazinas , Qualidade de Vida , Receptor ErbB-2 , Feminino , Humanos , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Fadiga/induzido quimicamente , Mutação , Náusea , Medidas de Resultados Relatados pelo Paciente , VômitoRESUMO
Cortical actin dynamics shapes cells. To generate actin filaments, cells rely on actin nucleators. Cobl is a novel, brain-enriched, WH2 domain-based actin nucleator, yet, its functions remained largely elusive. Here, we reveal that Cobl plays a crucial role in Purkinje cell development using gene gun transfections within intact murine cerebellar contexts. Cobl deficiency impaired proper dendritic arborization of Purkinje cells and led to low-complexity arbors. Branch point numbers and density and especially higher order branching were strongly affected. Our efforts to reveal how Cobl is physically and functionally integrated into the cortical actin cytoskeleton showed that all Cobl loss-of-function phenotypes were exactly mirrored by knockdown of the F-actin-binding protein Abp1. By subcellular fractionations, protein interaction analyses, subcellular reconstitutions of protein complexes, colocalization studies in cells and tissues, and by functional analyses in neuronal morphogenesis we demonstrate that both proteins associate and work with each other closely. Cobl-mediated dendritic branch induction in hippocampal neurons critically relied on Abp1. Our study highlights that the functions of Abp1 are distinct from those of the Cobl-binding protein syndapin I. The importance of Cobl/Abp1 complex formation and of Abp1-mediated F-actin association was highlighted by functional rescue experiments demonstrating that a Cobl mutant deficient for Abp1 binding and an Abp1 mutant supporting Cobl association but lacking the F-actin binding ability failed to rescue the respective loss-of-function phenotypes. Thus, F-actin-anchored Cobl/Abp1 complexes seem crucial for neuromorphogenesis processes, particularly for the postnatal arborization of Purkinje cells representing the source for all motor coordination in the cerebellar cortex.
Assuntos
Cerebelo/crescimento & desenvolvimento , Proteínas dos Microfilamentos/fisiologia , Neurogênese/fisiologia , Proteínas/fisiologia , Células de Purkinje/fisiologia , Domínios de Homologia de src/fisiologia , Actinas/metabolismo , Animais , Cerebelo/metabolismo , Proteínas do Citoesqueleto , Dendritos/ultraestrutura , Técnicas de Silenciamento de Genes/métodos , Hipocampo/citologia , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Imagem Molecular/métodos , Mutação , Ligação Proteica , Proteínas/genética , Proteínas/metabolismo , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Transfecção/métodos , Domínios de Homologia de src/genéticaRESUMO
Previous studies in our laboratory have shown that in models for three distinct forms of the inherited and incurable nerve disorder, Charcot-Marie-Tooth neuropathy, low-grade inflammation implicating phagocytosing macrophages mediates demyelination and perturbation of axons. In the present study, we focus on colony-stimulating factor-1, a cytokine implicated in macrophage differentiation, activation and proliferation and fostering neural damage in a model for Charcot-Marie-Tooth neuropathy 1B. By crossbreeding a model for the X-linked form of Charcot-Marie-Tooth neuropathy with osteopetrotic mice, a spontaneous null mutant for colony-stimulating factor-1, we demonstrate a robust and persistent amelioration of demyelination and axon perturbation. Furthermore, functionally important domains of the peripheral nervous system, such as juxtaparanodes and presynaptic terminals, were preserved in the absence of colony-stimulating factor-1-dependent macrophage activation. As opposed to other Schwann cell-derived cytokines, colony-stimulating factor-1 is expressed by endoneurial fibroblasts, as revealed by in situ hybridization, immunocytochemistry and detection of ß-galactosidase expression driven by the colony-stimulating factor-1 promoter. By both light and electron microscopic studies, we detected extended cell-cell contacts between the colony-stimulating factor-1-expressing fibroblasts and endoneurial macrophages as a putative prerequisite for the effective and constant activation of macrophages by fibroblasts in the chronically diseased nerve. Interestingly, in human biopsies from patients with Charcot-Marie-Tooth type 1, we also found frequent cell-cell contacts between macrophages and endoneurial fibroblasts and identified the latter as main source for colony-stimulating factor-1. Therefore, our study provides strong evidence for a similarly pathogenic role of colony-stimulating factor-1 in genetically mediated demyelination in mice and Charcot-Marie-Tooth type 1 disease in humans. Thus, colony-stimulating factor-1 or its cognate receptor are promising target molecules for treating the detrimental, low-grade inflammation of several inherited neuropathies in humans.
Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Neurônios/metabolismo , Animais , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Conexinas/genética , Conexinas/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Ativação de Macrófagos , Fator Estimulador de Colônias de Macrófagos/genética , Macrófagos/patologia , Camundongos , Neurônios/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nervo Sural/metabolismo , Nervo Sural/patologia , Regulação para Cima , beta-Galactosidase/metabolismo , Proteína beta-1 de Junções ComunicantesRESUMO
Thyroid hormone (TH) transporter MCT8 deficiency causes severe locomotor disabilities likely due to insufficient TH transport across brain barriers and, consequently, compromised neural TH action. As an established animal model for this disease, Mct8/Oatp1c1 double knockout (DKO) mice exhibit strong central TH deprivation, locomotor impairments and similar histo-morphological features as seen in MCT8 patients. The pathways that cause these neuro-motor symptoms are poorly understood. In this paper, we performed proteome analysis of brain sections comprising cortical and striatal areas of 21-day-old WT and DKO mice. We detected over 2900 proteins by liquid chromatography mass spectrometry, 67 of which were significantly different between the genotypes. The comparison of the proteomic and published RNA-sequencing data showed a significant overlap between alterations in both datasets. In line with previous observations, DKO animals exhibited decreased myelin-associated protein expression and altered protein levels of well-established neuronal TH-regulated targets. As one intriguing new candidate, we unraveled and confirmed the reduced protein and mRNA expression of Pde10a, a striatal enzyme critically involved in dopamine receptor signaling, in DKO mice. As altered PDE10A activities are linked to dystonia, reduced basal ganglia PDE10A expression may represent a key pathogenic pathway underlying human MCT8 deficiency.
Assuntos
Proteoma , Simportadores , Animais , Humanos , Camundongos , Proteoma/metabolismo , Proteômica , Simportadores/genética , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Hormônios Tireóideos/metabolismo , Diester Fosfórico Hidrolases/metabolismoRESUMO
Luminal breast cancers are hormone receptor (estrogen and/or progesterone) positive that are further divided into HER2-negative luminal A and HER2-positive luminal B subtypes. According to currently accepted convention, they represent the most common subtypes of breast cancer, accounting for approximately 70% of cases. Biomarkers play a critical role in the functional characterization, prognostication, and therapeutic prediction, rendering them indispensable for the clinical management of invasive breast cancer. Traditional biomarkers include clinicopathological parameters, which are increasingly extended by genetic and other molecular markers, enabling the comprehensive characterization of patients with luminal breast cancer. Liquid biopsies capturing and analyzing circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are emerging technologies that envision personalized management through precision oncology. This article reviews key biomarkers in luminal breast cancer and ongoing developments.