Thyroid hormone homeostasis in the perinatal mouse brain: implications for MCT8 transport defect

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 30-01-2017Thyroid hormones (TH) play an essential role both in the developing and the adult central nervous system (CNS). The concentrations of the genomically active hormone T3 in the brain depend on TH transport across the brain barriers, the local generation of T3 in astrocytes by type 2 deiodinase (D2), and TH degradation by type 3 deiodinase (D3). In addition, during foetal development, circulating TH concentrations derive partly from the mother. How all these factors interact during mice foetal development to ensure “brain euthyroidism” is not well known. This work has explored the sources of T3 that ensure brain euthyroidism during mice foetal development by treating euthyroid pregnant dams with T4 or T3 from embryonic day 12 (E12) to E18. The outcome of the treatment revealed that a large amount of foetal T4 during late gestation is of maternal origin and that maternal-foetal T3 transfer is tightly regulated, most probably at the placental level. Also, that T4 transport across the choroid plexus along with D2 activity at the blood-cerebrospinal fluid barrier might be key events in TH action in brain during foetal development, as conversion of T4 into T3 by D2 activity might be the only source of T3 during mouse brain development. Mutations in the gene expressing the TH monocarboxylate transporter 8 (MCT8), in humans, lead to altered circulating TH levels and a severe neurodevelopmental disorder. Due to the crucial role of this transporter, the interaction between Mct8-dependent transport and D2 activity was studied. D2 activity was found to be required to generate the brain hyperthyroidism characteristic of Mct8 deficient (Mct8KO) animals during perinatal stages of development. Besides, Mct8KO mice were used to test novel therapeutic approaches as possible treatments for MCT8 deficiency. The use of the TH analogue triiodothyroacetic acid (TRIAC) restored plasma T3 levels but severely decreased T4 levels leading to a state of brain hypothyroidism with reduced T3 content. Delivery of MCT8 by adeno-associated virus vectors proved that, for a successful gene therapy, restoration of MCT8 at the brain barriers is critical to mediate TH access to the brain of MCT8 deficient patients. Finally, intranasal delivery of TH as an alternative administration route increased TH levels in the systemic circulation, aggravating the peripheral hyperthyroidism, suggesting that is not a treatment option for MCT8 deficient patients.Las hormonas tiroideas (HT) juegan un papel esencial en el desarrollo y función del sistema nervioso central. En el cerebro, las concentraciones de la hormona activa a nivel genómico, T3, dependen del transporte de HT a través de las barrera cerebrales, de la generación local de T3 en los astrocitos mediante la desyodasa tipo 2 (D2) y de la degradación de HT por la desyodasa tipo 3 (D3). Además, durante el desarrollo fetal las concentraciones de HT circulantes provienen en parte de la madre. Cómo interactúan todos estos factores durante el desarrollo fetal en ratón para asegurar un “eutiroidismo cerebral” no se conoce suficientemente. En este trabajo se han explorado las fuentes de T3 que posibilitan un eutiroidismo cerebral durante el desarrollo fetal del ratón mediante el tratamiento de madres gestantes con T4 o T3 desde el día embrionario 12 (E12) hasta E18. El resultado del tratamiento reveló que una gran parte de la T4 fetal durante las fases finales de la gestación es de origen materno y que la transferencia materno-fetal de T3 está estrictamente regulada, probablemente a nivel placentario. También, que el transporte de T4 a través del plexo coroideo junto con la actividad de D2 en la barrera sangre-líquido cefalorraquídeo podrían ser eventos claves en la acción de las HT en el cerebro durante el desarrollo fetal, ya que la conversión de T4 a T3 mediante la actividad de D2 podría ser la única fuente de T3 durante el desarrollo cerebral del ratón. Mutaciones en el gen que expresa el transportador de HT, transportador de monocarboxilatos 8 (MCT8), provocan alteraciones en los niveles circulantes de HT y discapacidades graves del neurodesarrollo en humanos. Debido al papel fundamental de este transportador, se estudió la interacción entre el transporte dependiente de Mct8 y la actividad de D2. Los resultados indican que la actividad de D2 es necesaria para generar el hipertiroidismo cerebral característico de los ratones deficientes de Mct8 durante etapas perinatales del desarrollo. Además, se utilizaron ratones deficientes de Mct8 para examinar nuevas aproximaciones terapéuticas como posibles tratamientos para la deficiencia de MCT8. El uso del análogo de HT ácido triyodotiroacético (TRIAC) restableció los niveles plasmáticos de T3 pero redujo drásticamente los niveles de T4 generando un estado de hipotiroidismo cerebral con bajo contenido de T3. La administración de MCT8 mediante virus adeno-asociados demostró que, para una eficaz terapia génica, es crítico reestablecer MCT8 en las barreras cerebrales para mediar el acceso de HT en el cerebro de pacientes deficientes de MCT8. Finalmente, la administración de HT por vía intranasal como vía alternativa aumentó los niveles circulantes de HT agravando el hipertiroidismo periférico lo que sugiere que no es una opción para el tratamiento de pacientes deficientes de MCT8

Thyroid Hormone Availability and Action during Brain Development in Rodents

Thyroid hormones (THs) play an essential role in the development of all vertebrates; in particular adequate TH content is crucial for proper neurodevelopment. TH availability and action in the brain are precisely regulated by several mechanisms, including the secretion of THs by the thyroid gland, the transport of THs to the brain and neural cells, THs activation and inactivation by the metabolic enzymes deiodinases and, in the fetus, transplacental passage of maternal THs. Although these mechanisms have been extensively studied in rats, in the last decade, models of genetically modified mice have been more frequently used to understand the role of the main proteins involved in TH signaling in health and disease. Despite this, there is little knowledge about the mechanisms underlying THs availability in the mouse brain. This mini-review article gathers information from findings in rats, and the latest findings in mice regarding the ontogeny of TH action and the sources of THs to the brain, with special focus on neurodevelopmental stages. Unraveling TH economy and action in the mouse brain may help to better understand the physiology and pathophysiology of TH signaling in brain and may contribute to addressing the neurological alterations due to hypo and hyperthyroidism and TH resistance syndromes

Intracerebroventricular administration of the thyroid hormone analog TRIAC increases its brain content in the absence of MCT8

Patients lacking the thyroid hormone (TH) transporter MCT8 present abnormal serum levels of TH: low thyroxine and high triiodothyronine. They also have severe neurodevelopmental defects resulting from cerebral hypothyroidism, most likely due to impaired TH transport across the brain barriers. The use of TH analogs, such as triiodothyroacetic acid (TRIAC), that can potentially access the brain in the absence of MCT8 and restore at least a subset of cerebral TH actions could improve the neurological defects in these patients. We hypothesized that direct administration of TRIAC into the brain by intracerebroventricular delivery to mice lacking MCT8 could bypass the restriction at the brain barriers and mediate TH action without causing hypermetabolism. We found that intracerebroventricular administration of therapeutic doses of TRIAC does not increase further plasma triiodothyronine or further decrease plasma thyroxine levels and does not alter TH content in the cerebral cortex. Although TRIAC content increased in the brain, it did not induce TH-mediated actions on selected target genes. Our data suggest that intracerebroventricular delivery of TRIAC has the ability to target the brain in the absence of MCT8 and should be further investigated to address its potential therapeutic use in MCT8 deficiency.This work was funded by the Spanish Plan Nacional de I+D+i (grant number SAF2017-86342-R to AG-F), the Sherman Foundation (OTR02211 to AG-F and SB-L), the Center for Biomedical Research on Rare Diseases (Ciberer to AG-F and CG-M), Instituto de Salud Carlos III, Madrid, Spain. X-HL and SR were supported in part by grant DK 15070 from the National Institutes of Health, USA

MCT8 deficiency: The road to therapies for a rare disease

Allan-Herndon-Dudley syndrome is a rare disease caused by inactivating mutations in the SLC16A2 gene, which encodes the monocarboxylate transporter 8 (MCT8), a transmembrane transporter specific for thyroid hormones (T3 and T4). Lack of MCT8 function produces serious neurological disturbances, most likely due to impaired transport of thyroid hormones across brain barriers during development resulting in severe brain hypothyroidism. Patients also suffer from thyrotoxicity in other organs due to the presence of a high concentration of T3 in the serum. An effective therapeutic strategy should restore thyroid hormone serum levels (both T3 and T4) and should address MCT8 transporter deficiency in brain barriers and neural cells, to enable the access of thyroid hormones to target neural cells. Unfortunately, targeted therapeutic options are currently scarce and their effect is limited to an improvement in the thyrotoxic state, with no sign of any neurological improvement. The use of thyroid hormone analogs such as TRIAC, DITPA, or sobetirome, that do not require MCT8 to cross cell membranes and whose controlled thyromimetic activity could potentially restore the normal function of the affected organs, are being explored to improve the cerebral availability of these analogs. Other strategies aiming to restore the transport of THs through MCT8 at the brain barriers and the cellular membranes include gene replacement therapy and the use of pharmacological chaperones. The design of an appropriate therapeutic strategy in combination with an early diagnosis (at prenatal stages), will be key aspects to improve the devastating alterations present in these patients.This work was supported by the Spanish Ministry of Economy and Competitiveness, grant number SAF2017-86342-R (MINECO/AEI/FEDER, UE) to AG-F, the Sherman Foundation (Grant Number OTR02211) to AG-F and SB-L, and the BBSRC (Grant Number BB/R016879/1) to SB-L. CG-M is a recipient of a contract from the Center for Biomedical Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid. The cost of this publication has been paid in part by FEDER funds

Deficient thyroid hormone transport to the brain leads to impairments in axonal caliber and oligodendroglial development

Mutations in the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) lead to profound brain alterations, including myelination impairments, in humans. We aimed to further explore the pathophysiological mechanisms underlying the MCT8 deficiency-associated myelination impairments to unravel new biomarkers and therapeutic targets. We have performed brain histological analysis on an MCT8-deficient subject and histological, ultrastructural, and magnetic resonance imaging (MRI) analysis in the brain of a mouse model of the syndrome, lacking MCT8 and enzyme deiodinase type 2 (DIO2, Mct8/Dio2 KO). We have found that the MCT8-deficient subject presents severely reduced myelin lipid and protein staining and increased proportion of small-caliber myelinated axons in detriment of large-caliber ones. Mct8/Dio2 KO mice present myelination impairments and abnormal oligodendroglial development. We conclude that the greater proportion of small-caliber axons and impairments in the oligodendroglia lineage progression arise as potential mechanisms underlying the permanent myelination defects in MCT8-deficiency. Moreover, we present the Mct8/Dio2 KO mouse model, and MRI as a non-invasive biomarker, as highly valuable tools for preclinical studies involving MCT8 deficiency. These findings contribute to the understanding of the pathological mechanisms in MCT8 deficiency and suggest new biomarkers and therapeutic targets to consider therapeutic options for the neurological defects in patients.This study was supported by MCIN/AEI/10.13039/501100011033, Spain (Grant No. SAF2017-86342-R to AG-F); MCIN/AEI/10.13039/501100011033/FEDER “Una manera de hacer Europa”, Spain (Grant No PID2020-113139RB-I00 to AG-F); Consejo Superior de Investigaciones Científicas, Spain (Grant No. 2020AEP044 to AG-F); the Sherman Foundation, Australia (Grant No. OTR02211 to SB-L and AG-F); Asociación Corriendo con el Corazón por Hugo, Spain (Grant No OTR06190 to AG-F), and the BBSRC, United Kingdom (grant number BB/R016879/1 to SB-L). VV-H is recipient of a contract from MCIN/AEI /10.13039/501100011033/FSE “El FSE invierte en tu futuro”, Spain (Grant No PRE2018-086185), MG-Y from the programa de Formación de Profesorado Universitario, Spain (FPU, FPU19/02006) program from the Ministerio de Ciencia, Innovación y Universidades and DL-E was recipient of a fellowship from the “Fellowship Training Program for Advanced Human Capital, Becas Chile” from National Commission for Scientific and Technological Research (CONICYT), Gobierno de Chile

Intranasal delivery of Thyroid hormones in MCT8 deficiency

Loss of function mutations in the gene encoding the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) lead to severe neurodevelopmental defects in humans associated with a specific thyroid hormone phenotype manifesting high serum 3,5,3’-triiodothyronine (T3) and low thyroxine (T4) levels. Patients present a paradoxical state of peripheral hyperthyroidism and brain hypothyroidism, this last one most likely arising from impaired thyroid hormone transport across the brain barriers. The administration of thyroid hormones by delivery pathways that bypass the brain barriers, such as the intranasal delivery route, offers the possibility to improve the neurological defects of MCT8-deficient patients. In this study, the thyroid hormones T4 and T3 were administrated intranasally in different mouse models of MCT8 deficiency. We have found that, under the present formulation, intranasal administration of thyroid hormones does not increase the content of thyroid hormones in the brain and further raises the peripheral thyroid hormone levels. Our data suggests intranasal delivery of thyroid hormones is not a suitable therapeutic strategy for MCT8 deficiency, although alternative formulations could be considered in the future to improve the nose-to-brain transport.This work was supported by the Spanish Ministry of Economy and Competitiveness, grant number SAF2017-86342-R (MINECO/AEI/FEDER, UE) to AG-F, the Sherman Foundation (grant number OTR02211) to AG-F and SB-L, and the BBSRC (grant number BB/R016879/1) to SB-L. CG-M is a recipient of a contract from the Center for Biomedical Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Madrid. S.R. was supported by grant DK15079 from the National Institutes of Health, USA. The cost of this publication has been paid in part by FEDER funds (European Funds for Regional Development). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

Hormonas tiroideas y sistema nervioso central: implicaciones fisiológicas y fisiopatológicas

Trabajo presentado a la XX Jornada Científica del IIBM (Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM) celebrada el 23 de noviembre de 2017.Peer reviewe

AAV9-based gene therapy delivers a functional MCT8 transporter which improves thyroid hormone availability to brain of Mct8 deficient mice

Resumen del trabajo presentado al MCT8 Symposium: "Current Knowledge, Future Research on Treatment", celebrado en California (USA) del 12 al 14 de enero de 2016.-- et al.MCT8 gene mutations produce thyroid hormone (TH) deficiency in brain, causing severe neuropsychomotor abnormalities not correctable by TH treatment. We examined whether transfer of human MCT8 (hMCT8) cDNA using adeno-associated virus 9 (AAV9) could correct the brain defects of Mct8 knockout mice (Mct8KO). AAV9-hMCT8 or empty vector were injected intraventricularly (IV) and/or intracerebroventricularly (ICV) into postnatal day 1 Mct8KO mice and the active TH, T3, was given for 4 days before tissue collection at post-natal day 28. Compared to IV, ICV delivery produced more hMCT8 mRNA and protein, which was present in various brain regions and localized to the cell membranes. Despite more abundant hMCT8 mRNA and protein with ICV delivery, only IV delivered AAV9-hMCT8 targeted the choroid plexus and significantly increased brain T3 content and expression of the TH-regulated transcription factor, Hairless. These results indicate that MCT8 delivery to brain barriers by IV but not ICV injection is crucial for its proper function. MCT8 has no constitutive activity but acts through an increase in T3 entering brain tissue. The correct hMCT8 isoform along with an optimized delivery method are critical for an effective gene therapy to provide functional MCT8 in the brain of patients with MCT8 mutations.Peer Reviewe