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

Innovaciones y mejoras en el proyecto tutoría entre compañeros. Curso 2015-2016

Memoria ID-0137. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2015-2016

Monocarboxylate 8 transporter and deiodinase 2 deficiency impairs neurogliogesis in the adult mouse subventricular zone leading to cellular and functional alterations

Resumen del trabajo presentado en 44th Annual Meeting of the European Thyroid Association, celebrado en Bruselas (Bélgica) del 10 al 13 de septiembre de 2022.Thyroid hormones (THs) play a crucial role orchestrating neurodevelopment, but also regulate adult brain function. Recently, the potent effects that THs exert in adult neurogenic niches have started to be uncovered in rodents. These include an important role in the modulation of progenitor generation, especially controlling whether a neural stem cell (NSC) determines to become a neuronal or an oligodendroglial progenitor in the adult subventricular zone (SVZ), the largest NSC niche in the mammalian brain. A complex network of regulators tightly modulates TH availability and action, including transmembrane transporters, deiodinases and receptors. Among the TH-transporters, there is only one that is TH-specific, the monocarboxylate transporter 8 (MCT8). Deficiency of MCT8 leads to an ultra-rare but devastating disease, the Allan-Herndon-Dudley Syndrome (AHDS). Patients exhibit a plethora of endocrine and severe neurological disturbances and so far, no effective treatment for their neurological symptoms exists. Its complexity, along with its low prevalence and severe symptomatology, makes animal models and biomarkers of the disease a crucial step in the research for potential strategies to alleviate the patients’ severe conditions. Using a well-validated animal model of AHDS, the Mct8/Dio2 KO mice, we aimed to characterize how a reduced T3 availability structurally and functionally affected the neurogenic and gliogenic capacity of the adult SVZ-NSCs. To this end, we analysed the expression of cell markers by immunohistochemistry to study the balance between neurons and glia in the SVZ, both in-vivo and using ex-vivo neurosphere cultures. These studies revealed severe alterations in the neuroglial balance, with an increase of the neuron/glia ratio in the SVZ in adult Mct8/Dio2 KO mice. We also observed that MCT8/DIO2 deficiency reduced NSC proliferation two-fold and hampered migrating of proliferating neuronal progenitors. Moreover, we tested the effects of administering exogenous THs and TH-analogues on neurospheres prepared from dissected SVZs. Neither the neuron/glia balance, nor proliferative activity responded to TH treatment in MCT8/DIO2 deficient neurospheres. Also, behaviour consequences of the observed NSCs alterations were studied using the olfactory memory and odour discrimination tests, as potential non-invasive biomarkers of the disease. These tests revealed that Mct8/Dio2 KO mice did not recognize new odours and failed to memorize them. Altogether, these results indicate that MCT8/DIO2 deficiency severely hampers TH-dependent regulation of adult SVZ-neurogliogenesis and suggest potential biomarkers for future preclinical studies

Blood-brain barrier disruption and increased permeability in mice deficient in the thyroid hormone transporter MCT8

Trabajo presentado en el 24th International Symposium on Signal Transduction at the Blood-Brain Barriers, celebrado en Bari (Italia) del 21 al 23 de septiembre de 2022.[Introduction]: The monocarboxylate transporter 8 (MCT8) is crucial for the maintenance of thyroid hormones (TH) homeostasis in the brain, and it is the only TH transporter known to date that exclusively transports TH across the blood-brain barrier (BBB) and into the TH target cells. The Allan-Herndon-Dudley Syndrome (AHDS), or MCT8 deficiency, is an ultrarare X-linked disease due to inactivating mutations in the MCT8 gene (SLC16A2). Patients show delayed neurodevelopment and severe psychomotor disorders, most probably due to impaired transport of TH across the BBB, although the pathological mechanisms are poorly understood; with no effective treatments for the neurological symptoms of patients. [Aims]: To assess the BBB integrity, permeability and ultrastructure in an animal model of MCT8-deficiency: the Mct8/Dio2KO mouse. [Methods]: We used transmission electron microscopy to evaluate neurovascular unit (NVU) components and western blot to evaluate the expression of NVU and extracellular matrix-related proteins. BBB functionality was assessed by analyzing brain infiltration of non-permeable dyes and quantifying parenchymal microhemorrhages. Finally, the whole-brain blood vessel density was studied by magnetic resonance angiography. [Results]: Ultrastructural analysis revealed significant alterations in NVU integrity and signs of increased transcytotic flux, along with increased permeability of the BBB, increased presence of brain microhemorrhages, and a significant decrease in blood vessel density in Mct8/Dio2KO mice. Discussion. Our results provide a novel paradigm for AHDS, which can result in potential new therapeutic targets and biomarkers associated with the neurological symptoms of the disease and contribute to the generation of therapeutic strategies to improve MCT8-deficient patients¿ life qualit

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

[Background]: Monocarboxylate transporter 8 (MCT8) deficiency is a rare X-linked disease where patients exhibit peripheral hyperthyroidism and cerebral hypothyroidism, which results in severe neurological impairments. These brain defects arise from a lack of thyroid hormones (TH) during critical stages of human brain development. Treatment options for MCT8-deficient patients are limited and none have been able to prevent or ameliorate effectively the neurological impairments. This study explored the effects of the TH agonist sobetirome and its CNS-selective amide prodrug, Sob-AM2, in the treatment of pregnant dams carrying fetuses lacking Mct8 and deiodinase type 2 (Mct8/Dio2 KO), as a murine model for MCT8 deficiency. [Methods]: Pregnant dams carrying Mct8/Dio2 KO fetuses were treated with 1 mg of sobetirome/kg body weight/day, or 0.3 mg of Sob-AM2/kg body weight/day for 7 days, starting at embryonic day 12.5 (E12.5). As controls, pregnant dams carrying wild-type and pregnant dams carrying Mct8/Dio2 KO fetuses were treated with daily subcutaneous injections of vehicle. Dams TH levels were measured by enzyme-linked immunosorbent assay (ELISA). Samples were extracted at E18.5 and the effect of treatments on the expression of triiodothyronine (T3)-dependent genes was measured in the placenta, fetal liver, and fetal cerebral cortex by real-time polymerase chain reaction. [Results]: Maternal sobetirome treatment led to spontaneous abortions. Sob-AM2 treatment, however, was able to cross the placental as well as the brain barriers and exert thyromimetic effects in Mct8/Dio2 KO fetal tissues. Sob-AM2 treatment did not affect the expression of the T3-target genes analyzed in the placenta, but it mediated thyromimetic effects in the fetal liver by increasing the expression of Dio1 and Dio3 genes. Interestingly, Sob-AM2 treatment increased the expression of several T3-dependent genes in the brain such as Hr, Shh, Dio3, Kcnj10, Klf9, and Faah in Mct8/Dio2 KO fetuses. [Conclusions]: Maternal administration of Sob-AM2 can cross the placental barrier and access the fetal tissues, including the brain, in the absence of MCT8, to exert thyromimetic actions by modulating the expression of T3-dependent genes. Therefore, Sob-AM2 has the potential to address the cerebral hypothyroidism characteristic of MCT8 deficiency from fetal stages and to prevent neurodevelopmental alterations in the MCT8-deficient fetal brain.This study was supported by the Spanish Ministry of Science and Innovation (MCIN)/AEI/10.13039/501100011033 (grant no. SAF2017-86342-R to A.G.-F.); MCIN/AEI/10.13039/501100011033/FEDER ‘‘Una manera de hacer Europa’’ (grant no. PID2020-113139RB-I00 to A.G.-F.); Consejo Superior de Investigaciones Cientı´ficas (grant no. 2020AEP044 to A.G.-F.); the Sherman Foundation (grant no. OTR02211 to S.B.-L. and A.G.-F.); Asociacio´n Corriendo con el Corazo´n por Hugo (grant no. OTR06190 to A.G.-F.), and MCIN/EU (grant no. IJC2020-043543-I) to S.B.-L. V.V.-H. is recipient of a contract from MCIN ‘‘El FSE invierte en tu futuro’’ (grant no. PRE2018-086185) and M.G.-Y. from the MCIN, Programa de Formacio´n de Profesorado Universitario (FPU, FPU19/02006).Peer reviewe

Neurological alterations and abnormal thyroid hormone levels/content in plasma and tissues in a CRISPR/Cas9-engineered avatar mouse model of monocarboxylate transporter 8 deficiency

Trabajo presentado en MCT8 Symposium, celebrado en Bruselas (Bélgica) el 08 de septiembre de 2022.An optimal mammalian model to explore the pathological mechanisms and potential therapeutic approaches is essential to improve the well-being of MCT8-deficient patients. The absence of neurological defects in single Mct8 KO mice seems to result from a compensatory mechanism in mice involving the T4 transport through the Organic Anion Transporter 1C1 (OATP1C1) across the brain barriers and the local conversion of T4 into T3 by the enzyme deiodinase 2 (DIO2) on the astrocytes. For this, the double mutant mice Mct8/Oatp1c1 KO and Mct8/Dio2 KO display neurodevelopmental defects and are being used as disease animal models for MCT8 deficiency. Here we have generated by CRISPR/Cas9 an avatar mouse model for MCT8 deficiency with a point mutation found in two MCT8- deficient patients (P321L, P253L in mice). These mice provide a unique opportunity to explore the consequences of the P253L substitution, instead of deleting MCT8 alone or inactivating both MCT8 and OATP1C1 or DIO2. We have predicted by in silico studies that this mutation alters the substrate binding pocket impairing thyroid hormone transport. We have also characterized the phenotype of P253L mice and found endocrine alterations similar to those described in patients, including brain hypothyroidism and peripheral hyperthyroidism as well as structural and functional neurological alterations resembling the patient´s neurological impairments. We predict that the P253L mouse model will provide further understanding regarding the pathophysiology of MCT8 deficiency and will allow testing of therapeutic alternatives such as in vivo gene therapy and pharmacological chaperone therapy to improve the neurological impairments in MCT8 deficiency

Neurovascular unit disruption and blood–brain barrier leakage in MCT8 deficiency

[Background]: The monocarboxylate transporter 8 (MCT8) plays a vital role in maintaining brain thyroid hormone homeostasis. This transmembrane transporter is expressed at the brain barriers, as the blood–brain barrier (BBB), and in neural cells, being the sole known thyroid hormone-specific transporter to date. Inactivating mutations in the MCT8 gene (SLC16A2) cause the Allan-Herndon-Dudley Syndrome (AHDS) or MCT8 deficiency, a rare X-linked disease characterized by delayed neurodevelopment and severe psychomotor disorders. The underlying pathophysiological mechanisms of AHDS remain unclear, and no effective treatments are available for the neurological symptoms of the disease.[Methods]: Neurovascular unit ultrastructure was studied by means of transmission electron microscopy. BBB permeability and integrity were evaluated by immunohistochemistry, non-permeable dye infiltration assays and histological staining techniques. Brain blood-vessel density was evaluated by immunofluorescence and magnetic resonance angiography. Finally, angiogenic-related factors expression was evaluated by qRT-PCR. The studies were carried out both in an MCT8 deficient subject and Mct8/Dio2KO mice, an AHDS murine model, and their respective controls.[Results]: Ultrastructural analysis of the BBB of Mct8/Dio2KO mice revealed significant alterations in neurovascular unit integrity and increased transcytotic flux. We also found functional alterations in the BBB permeability, as shown by an increased presence of peripheral IgG, Sodium Fluorescein and Evans Blue, along with increased brain microhemorrhages. We also observed alterations in the angiogenic process, with reduced blood vessel density in adult mice brain and altered expression of angiogenesis-related factors during brain development. Similarly, AHDS human brain samples showed increased BBB permeability to IgG and decreased blood vessel density.[Conclusions]: These findings identify for the first time neurovascular alterations in the MCT8-deficient brain, including a disruption of the integrity of the BBB and alterations in the neurovascular unit ultrastructure as a new pathophysiological mechanism for AHDS. These results open a new field for potential therapeutic targets for the neurological symptoms of these patients and unveils magnetic resonance angiography as a new non-invasive in vivo technique for evaluating the progression of the disease.This study was supported by grants from the Spanish Ministry of Research and Innovation MCIN/AEI/10. 13039/501100011033, and by “ERDF A way of making Europe” (Grants No. SAF2017-86342-R and 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 AG-F); Asociación Corriendo con el Corazón por Hugo, Spain (Grant No. OTR06190 to AG-F) and Fundación Ciudad de Alcobendas and Alcobendas City Council (Donation code OTR08490 to AG-F). MG-Y is a recipient of a fellowship from the Programa de Formación de Profesorado (FPU, FPU19/02006), Plan Estatal de I + D + I of the Ministerio de Universidades, AG-A is a recipient of a contract from UAM (FPI) and VV-H is a recipient of a contract from MCIN/AEI/10. 13039/501100011033 and by “ESF Investing in your future”, Spain (Grant No. PRE2018-086185).Peer reviewe

Neurovascular unit disruption and blood–brain barrier leakage in MCT8 deficiency

Abstract Background The monocarboxylate transporter 8 (MCT8) plays a vital role in maintaining brain thyroid hormone homeostasis. This transmembrane transporter is expressed at the brain barriers, as the blood–brain barrier (BBB), and in neural cells, being the sole known thyroid hormone-specific transporter to date. Inactivating mutations in the MCT8 gene (SLC16A2) cause the Allan-Herndon-Dudley Syndrome (AHDS) or MCT8 deficiency, a rare X-linked disease characterized by delayed neurodevelopment and severe psychomotor disorders. The underlying pathophysiological mechanisms of AHDS remain unclear, and no effective treatments are available for the neurological symptoms of the disease. Methods Neurovascular unit ultrastructure was studied by means of transmission electron microscopy. BBB permeability and integrity were evaluated by immunohistochemistry, non-permeable dye infiltration assays and histological staining techniques. Brain blood-vessel density was evaluated by immunofluorescence and magnetic resonance angiography. Finally, angiogenic-related factors expression was evaluated by qRT-PCR. The studies were carried out both in an MCT8 deficient subject and Mct8/Dio2KO mice, an AHDS murine model, and their respective controls. Results Ultrastructural analysis of the BBB of Mct8/Dio2KO mice revealed significant alterations in neurovascular unit integrity and increased transcytotic flux. We also found functional alterations in the BBB permeability, as shown by an increased presence of peripheral IgG, Sodium Fluorescein and Evans Blue, along with increased brain microhemorrhages. We also observed alterations in the angiogenic process, with reduced blood vessel density in adult mice brain and altered expression of angiogenesis-related factors during brain development. Similarly, AHDS human brain samples showed increased BBB permeability to IgG and decreased blood vessel density. Conclusions These findings identify for the first time neurovascular alterations in the MCT8-deficient brain, including a disruption of the integrity of the BBB and alterations in the neurovascular unit ultrastructure as a new pathophysiological mechanism for AHDS. These results open a new field for potential therapeutic targets for the neurological symptoms of these patients and unveils magnetic resonance angiography as a new non-invasive in vivo technique for evaluating the progression of the disease