AAV9-MCT8 Delivery at Juvenile Stage Ameliorates Neurological and Behavioral Deficits in a Mouse Model of MCT8-Deficiency.

Background: Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor disability disorder that also manifests characteristic abnormal thyroid hormone (TH) levels. AHDS is caused by inactivating mutations in monocarboxylate transporter 8 (MCT8), a specific TH plasma membrane transporter widely expressed in the central nervous system (CNS). MCT8 mutations cause impaired transport of TH across brain barriers, leading to insufficient neural TH supply. There is currently no successful therapy for the neurological symptoms. Earlier work has shown that intravenous (IV), but not intracerebroventricular adeno-associated virus serotype 9 (AAV9) -based gene therapy given to newborn Mct8 knockout (Mct8-/y) male mice increased triiodothyronine (T3) brain content and partially rescued TH-dependent gene expression, suggesting a promising approach to treat this neurological disorder. Methods: The potential of IV delivery of AAV9 carrying human MCT8 was tested in the well-established Mct8-/y/Organic anion-transporting polypeptide 1c1 (Oatp1c1)-/ - double knockout (dKO) mouse model of AHDS, which, unlike Mct8-/y mice, displays both neurological and TH phenotype. Further, as the condition is usually diagnosed during childhood, treatment was given intravenously to P30 mice and psychomotor tests were carried out blindly at P120-P140 after which tissues were collected and analyzed. Results: Systemic IV delivery of AAV9-MCT8 at a juvenile stage led to improved locomotor and cognitive functions at P120-P140, which was accompanied by a near normalization of T3 content and an increased response of positively regulated TH-dependent gene expression in different brain regions examined (thalamus, hippocampus, and parietal cortex). The effects on serum TH concentrations and peripheral tissues were less pronounced, showing only improvement in the serum T3/reverse T3 (rT3) ratio and in liver deiodinase 1 expression. Conclusion: IV administration of AAV9, carrying the human MCT8, to juvenile dKO mice manifesting AHDS has long-term beneficial effects, predominantly on the CNS. This preclinical study indicates that this gene therapy has the potential to ameliorate the devastating neurological symptoms in patients with AHDS.

MPC1 Deficiency Promotes CRC Liver Metastasis via Facilitating Nuclear Translocation of ß-Catenin.

Accumulating evidence has pointed out that metastasis is the leading cause of death in several malignant tumor, including CRC. During CRC, metastatic capacity is closely correlated with reprogrammed energy metabolism. Mitochondrial Pyruvate Carrier 1 (MPC1), as the carrier of transporting pyruvate into mitochondria, linked the glycolysis and TCA cycle, which would affect the energy production. However, the specific role of MPC1 on tumor metastasis in CRC remains unexplored. Here, by data mining of genes involved in pyruvate metabolism using the TCGA dataset, we found that MPC1 was significantly downregulated in CRC compared to nontumor tissues. Similar MPC1 expression pattern was also found in multiple GEO datasets. IHC staining in both human sample and AOM/DSS induced mouse CRC model revealed significant downregulation of MPC1. What is more, we found that MPC1 expression was gradually decreased in normal tissue, primary CRC, and metastasis CRC. Additionally, poor prognosis emerged in the MPC1 low expression patients, especially in patients with metastasis. Following, functional tests showed that MPC1 overexpression inhibited the motility of CRC cells in vitro and MPC1 silencing enhanced liver metastases in vivo. Furthermore, we uncovered that decreased MPC1 activated the Wnt/ß-catenin pathway by promoting nuclear translocation of ß-catenin to mediate the expression of MMP7, E-cadherin, Snail1, and myc. Collectively, our data suggest that MPC1 has the potential to be served as a promising biomarker for diagnosis and a therapeutic target in CRC.

Mitochondrial Pyruvate Carrier 1 Promotes Peripheral T Cell Homeostasis through Metabolic Regulation of Thymic Development.

Metabolic pathways regulate T cell development and function, but many remain understudied. Recently, the mitochondrial pyruvate carrier (MPC) was identified as the transporter that mediates pyruvate entry into mitochondria, promoting pyruvate oxidation. Here we find that deleting Mpc1, an obligate MPC subunit, in the hematopoietic system results in a specific reduction in peripheral αß T cell numbers. MPC1-deficient T cells have defective thymic development at the ß-selection, intermediate single positive (ISP)-to-double-positive (DP), and positive selection steps. We find that early thymocytes deficient in MPC1 display alterations to multiple pathways involved in T cell development. This results in preferred escape of more activated T cells. Finally, mice with hematopoietic deletion of Mpc1 are more susceptible to experimental autoimmune encephalomyelitis. Altogether, our study demonstrates that pyruvate oxidation by T cell precursors is necessary for optimal αß T cell development and that its deficiency results in reduced but activated peripheral T cell populations.

Thyroid Hormone Transporters.

Thyroid hormone transporters at the plasma membrane govern intracellular bioavailability of thyroid hormone. Monocarboxylate transporter (MCT) 8 and MCT10, organic anion transporting polypeptide (OATP) 1C1, and SLC17A4 are currently known as transporters displaying the highest specificity toward thyroid hormones. Structure-function studies using homology modeling and mutational screens have led to better understanding of the molecular basis of thyroid hormone transport. Mutations in MCT8 and in OATP1C1 have been associated with clinical disorders. Different animal models have provided insight into the functional role of thyroid hormone transporters, in particular MCT8. Different treatment strategies for MCT8 deficiency have been explored, of which thyroid hormone analogue therapy is currently applied in patients. Future studies may reveal the identity of as-yet-undiscovered thyroid hormone transporters. Complementary studies employing animal and human models will provide further insight into the role of transporters in health and disease. (Endocrine Reviews 41: 1 - 55, 2020).

Roles of monocarboxylate transporter subtypes in promotion and suppression of osteoclast differentiation and survival on bone.

Monocarboxylate transporters (MCTs) provide transmembrane transport of monocarboxylates such as lactate and pyruvate. The present results showed that α-cyano-4-hydroxycinnamic acid (CHC), an inhibitor of MCTs, promoted osteoclast differentiation from macrophages at lower concentrations (0.1-0.3 mM) and suppressed that at a higher concentration (1.0 mM). On the other hand, CHC reduced the number of mature osteoclasts on the surface of dentin in a concentration-dependent manner. Additionally, macrophages and osteoclasts were found to express the Mct1, Mct2, and Mct4 genes, with Mct1 and Mct4 expression higher in macrophages, and that of Mct2 higher in osteoclasts. Although Mct1 gene knockdown in macrophages enhanced osteoclast formation induced by RANKL, Mct2 gene knockdown suppressed that. Finally, Mct2 gene silencing in mature osteoclasts decreased their number and, thereby, bone resorption. These results suggest that MCT1 is a negative regulator and MCT2 a positive regulator of osteoclast differentiation, while MCT2 is required for bone resorption by osteoclasts.

MPC1 deficiency accelerates lung adenocarcinoma progression through the STAT3 pathway.

Mitochondrial pyruvate carrier 1 (MPC1), a key factor that controls pyruvate transportation in the mitochondria, is known to be frequently dysregulated in tumor initiation and progression. However, the clinical relevance and potential molecular mechanisms of MPC1 in lung adenocarcinoma (LAC) progression remain to be illustrated. Herein, MPC1 was lowly expressed in LAC tissues and significantly associated with favorable survival of patients with LAC. Functionally, MPC1 markedly suppressed stemness, invasion, and migration in vitro and spreading growth of LAC cells in vivo. Further study revealed that MPC1 could interact with mitochondrial signal transducer and activator of transcription 3 (mito-STAT3), disrupting the distribution of STAT3 and reducing cytoplasmic signal transducer and activator of transcription 3 (cyto-STAT3) as well as its phosphorylation, while the activation of cyto-STAT3 by IL-6 reversed the attenuated malignant progression in MPC1-overexpression LAC cells. Collectively, we reveal that MPC1/STAT3 axis plays an important role in the progression of LAC, and our work may promote the development of new therapeutic strategies for LAC.

Branched-chain ketoacids secreted by glioblastoma cells via MCT1 modulate macrophage phenotype.

Elevated amino acid catabolism is common to many cancers. Here, we show that glioblastoma are excreting large amounts of branched-chain ketoacids (BCKAs), metabolites of branched-chain amino acid (BCAA) catabolism. We show that efflux of BCKAs, as well as pyruvate, is mediated by the monocarboxylate transporter 1 (MCT1) in glioblastoma. MCT1 locates in close proximity to BCKA-generating branched-chain amino acid transaminase 1, suggesting possible functional interaction of the proteins. Using in vitro models, we demonstrate that tumor-excreted BCKAs can be taken up and re-aminated to BCAAs by tumor-associated macrophages. Furthermore, exposure to BCKAs reduced the phagocytic activity of macrophages. This study provides further evidence for the eminent role of BCAA catabolism in glioblastoma by demonstrating that tumor-excreted BCKAs might have a direct role in tumor immune suppression. Our data further suggest that the anti-proliferative effects of MCT1 knockdown observed by others might be related to the blocked excretion of BCKAs.

Zebrafish - An emerging model to explore thyroid hormone transporters and psychomotor retardation.

Thyroid hormones (THs) regulate a variety of fundamental physiological processes, including the development and maintenance of the brain. For decades, it was thought that THs enter the cells by passive diffusion. However, it is now clear that TH transport across the cell membrane requires specific transporter proteins that facilitate the uptake and efflux of THs. Several thyroid hormone transmembrane transporters (THTTs) have been identified, including monocarboxylate transporter 8 (MCT8), MCT10, and organic anion transporting polypeptide 1C1 (OATP1C1). The critical role of THTTs in regulating metabolism and brain function is demonstrated in the Allan-Herndon-Dudley syndrome (AHDS), an X-linked psychomotor retardation associated with mutations in the MCT8/SLC16A2 gene. In addition to traditional research on humans, cell-lines, and rodents, the zebrafish has recently emerged as an attractive model to study THTTs and neuroendocrinological-related disorders. In this review, we describe the unique contribution of zebrafish studies to the understanding of the functional role of THTTs in live animals, and how this transparent vertebrate model can be used for translational studies on TH-related disorders.

[MCT8-specific thyroid hormone cell transporter deficiency: a case report and review of the literature]. / Deficiencia del transportador celular de hormona tiroidea MCT8: caso clinico y revision de la bibliografia.

INTRODUCTION: MCT8 is a specific transporter for the T4 and T3 thyroid hormones that allows their entry in the brain and other organs. Mutations in MCT8 (Allan-Herndon-Dudley syndrome) lead to a severe form of X-linked psychomotor retardation, which is characterised by elevated plasma T3 and low T4. AIM: We describe the first case diagnosed in Spain with this syndrome and review the published literature about this topic. We both review the various clinical presentations, genetic advances, differential diagnosis and therapeutic perspectives of this syndrome and propose a diagnostic algorithm for it. CASE REPORT: A 5 year-old boy, with a clinical picture compatible with Pelizaeus-Merzbacher disease. PLP1 gene sequencing showed no abnormalities. All the genetic and metabolic studies conducted were normal. Finally, a complete study of thyroid profile revealed abnormalities that were consistent with MCT8 transporter deficiency. The sequencing of the SLC16A2 gene (MCT8) showed a mutation in exon 3 and the study made at a cellular level, has confirmed that this mutation changes the properties of the protein. CONCLUSIONS: In the last five years, there have been many publications about this syndrome, with the identification of more than 50 families worldwide. It is important to both know and suspect this syndrome, because the diagnosis is easy, cheap and accessible (thyroid profile) and, although it has no specific treatment, early diagnosis prevents unnecessary testing and allows to offer genetic counseling to the families affected by it.

TITLE: Deficiencia del transportador celular de hormona tiroidea MCT8: caso clinico y revision de la bibliografia.Introduccion. El MCT8 es un transportador especifico para las hormonas tiroideas T4 y T3, que permite su entrada en el cerebro y otros organos. La deficiencia de MCT8, o sindrome de Allan-Herndon-Dudley, es un trastorno ligado a X que, generalmente, se presenta como un cuadro neurologico grave de inicio precoz, con un perfil tiroideo caracteristico (aumento de T3 y disminucion de T4 y rT3). Objetivo. Se presenta el primer caso diagnosticado en España con este sindrome y se revisa la bibliografia publicada, las distintas formas de presentacion clinica, los avances geneticos, el diagnostico diferencial y las perspectivas terapeuticas, y se propone un algoritmo diagnostico. Caso clinico. Varon de 5 años con un cuadro clinico compatible con una enfermedad de Pelizaeus-Merzbacher. La secuenciacion del gen PLP1 no mostro alteraciones. Todos los estudios metabolicos y geneticos realizados fueron normales. Finalmente, un estudio completo del perfil tiroideo revelo alteraciones compatibles con una deficiencia del transportador MCT8. La secuenciacion del gen SLC16A2 (MCT8) mostro una mutacion en el exon 3 y el estudio celular confirmo que esta mutacion cambia las propiedades de la proteina. Conclusiones. En los ultimos años se han multiplicado las publicaciones sobre este sindrome, con la identificacion de mas de 50 familias en el mundo. Es importante conocer este sindrome y sospecharlo, porque el diagnostico es facil, economico y accesible (perfil tiroideo), y, aunque no tiene tratamiento especifico, el diagnostico precoz evita pruebas innecesarias y permite ofrecer consejo genetico a las familias afectadas.

The pathophysiological consequences of thyroid hormone transporter deficiencies: Insights from mouse models.

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.

Monocarboxylate transporter 8 deficiency: altered thyroid morphology and persistent high triiodothyronine/thyroxine ratio after thyroidectomy.

CONTEXT: Thyroid hormone transport across the plasma membrane depends on transmembrane transport proteins, including monocarboxylate transporter 8 (MCT8). Mutations in MCT8 (or SLC16A2) lead to a severe form of X-linked psychomotor retardation, which is characterised by elevated plasma triiodothyronine (T(3)) and low/normal thyroxine (T(4)). MCT8 contributes to hormone release from the thyroid gland. OBJECTIVE: To characterise the potential impact of MCT8-deficiency on thyroid morphology in a patient and in Mct8-deficient mice. DESIGN: Thyroid morphology in a patient carrying the A224V mutation was followed by ultrasound imaging for over 10 years. After thyroidectomy, a histopathological analysis was carried out. The findings were compared with histological analyses of mouse thyroids from the Mct8(-/y) model. RESULTS: We show that an inactivating mutation in MCT8 leads to a unique, progressive thyroid follicular pathology in a patient. After thyroidectomy, histological analysis revealed gross morphological changes, including several hyperplastic nodules, microfollicular areas with stromal fibrosis and a small focus of microfollicular structures with nuclear features reminiscent of papillary thyroid carcinoma (PTC). These findings are supported by an Mct8-null mouse model in which we found massive papillary hyperplasia in 6- to 12-month-old mice and nuclear features consistent with PTC in almost 2-year-old animals. After complete thyroidectomy and substitution with levothyroxine (l-T(4)), the preoperative, inadequately low T(4) and free T(4) remained, while increasing the l-T(4) dosage led to T(3) serum concentrations above the normal range. CONCLUSIONS: Our results implicate peripheral deiodination in the peculiar hormonal constellation of MCT8-deficient patients. Other MCT8-deficient patients should be closely monitored for potential thyroid abnormalities.

RNA interference screen for human genes associated with West Nile virus infection.

West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus-host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV-human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.