From zebrafish to human: A comparative approach to elucidate the role of the thyroid hormone transporter MCT8 during brain development

Monocarboxylate transporter 8 (MCT8) facilitates transmembrane transport of thyroid hormones (THs) ensuring their action on gene expression during vertebrate neurodevelopment. A loss of MCT8 in humans results in severe psychomotor deficits associated with the Allan-Herndon-Dudley Syndrome (AHDS). However, where and when exactly a lack of MCT8 causes the neurological manifestations remains unclear because of the varying expression pattern of MCT8 between specific brain regions and cells. Here, we elaborate on the animal models that have been generated to elucidate the mechanisms underlying MCT8-deficient brain development. The absence of a clear neurological phenotype in Mct8 knockout mice made it clear that a single species would not suffice. The evolutionary conservation of TH action on neurodevelopment as well as the components regulating TH signalling however offers the opportunity to answer different aspects of MCT8 function in brain development using different vertebrate species. Moreover, the plethora of tools for genome editing available today facilitates gene silencing in these animals as well. Studies in the recently generated mct8-deficient zebrafish and Mct8/Oatp1c1 double knockout mice have put forward the current paradigm of impaired TH uptake at the level of the blood-brain barrier during peri- and postnatal development as being the main pathophysiological mechanism of AHDS. RNAi vector-based, cell-specific induction of MCT8 knockdown in the chicken embryo points to an additional function of MCT8 at the level of the neural progenitors during early brain development. Future studies including also additional in vivo models like Xenopus or in vitro approaches such as induced pluripotent stem cells will continue to help unravelling the exact role of MCT8 in developmental events. In the end, this multispecies approach will lead to a unifying thesis regarding the cellular and molecular mechanisms responsible for the neurological phenotype in AHDS patients.status: accepte

Dissecting the role of regulators of thyroid hormone availability in early brain development : merits and potential of the chicken embryo

Thyroid hormones (THs) are important mediators of vertebrate central nervous system (CNS) development, thereby regulating the expression of a wide variety of genes by binding to nuclear TH receptors. TH transporters and deiodinases are both needed to ensure appropriate intracellular TH availability, but the precise function of each of these regulators and their coaction during brain development is only partially understood. Rodent knockout models already provided some crucial insights, but their in utero development severely hampers research regarding the role of TH regulators during early embryonic stages. The establishment of novel gain- and loss-of-function techniques has boosted the position of externally developing non-mammalian vertebrates as research models in developmental endocrinology. Here, we elaborate on the chicken as a model organism to elucidate the function of TH regulators during embryonic CNS development. The fast-developing, relatively big and accessible embryo allows easy experimental manipulation, especially at early stages of brain development. Recent data on the characterisation and spatiotemporal expression pattern of different TH regulators in embryonic chicken CNS have provided the necessary background to dissect the function of each of them in more detail. We highlight some recent advances and important strategies to investigate the role of TH transporters and deiodinases in various CNS structures like the brain barriers, the cerebellum, the retina and the hypothalamus. Exploiting the advantages of this non-classical model can greatly contribute to complete our understanding of the regulation of TH bioavailability throughout embryonic CNS development.status: publishe

The importance of the thyroid hormone transporter MCT8 in the early embryonic development of the chicken retina

status: publishe

Deficiency of the thyroid hormone transporter Monocarboxylate Transporter 8 in neural progenitors impairs cellular processes crucial for early corticogenesis

Thyroid hormones (THs) are essential for establishing layered brain structures, a process called corticogenesis, by acting on transcriptional activity of numerous genes. In humans, deficiency of the monocarboxylate transporter 8 (MCT8), involved in cellular uptake of THs before their action, results in severe neurological abnormalities, known as the Allan-Herndon-Dudley syndrome. While the brain lesions predominantly originate prenatally, it remains unclear how and when exactly MCT8 dysfunction affects cellular processes crucial for corticogenesis. We investigated this by inducing in vivo RNAi vector-based knockdown of MCT8 in neural progenitors of the chicken optic tectum, a layered structure that shares many developmental features with the mammalian cerebral cortex. MCT8 knockdown resulted in cellular hypoplasia and a thinner optic tectum. This could be traced back to disrupted cell-cycle kinetics and a premature shift to asymmetric cell divisions impairing progenitor cell pool expansion. Birth-dating experiments confirmed diminished neurogenesis in the MCT8-deficient cell population as well as aberrant migration of both early-born and late-born neuroblasts, which could be linked to reduced reelin signaling and disorganized radial glial cell fibers. Impaired neurogenesis resulted in a reduced number of glutamatergic and GABAergic neurons, but the latter additionally showed decreased differentiation. Moreover, an accompanying reduction in untransfected GABAergic neurons suggests hampered intercellular communication. These results indicate that MCT8-dependent TH uptake in the neural progenitors is essential for early events in corticogenesis, and help to understand the origin of the problems in cortical development and function in Allan-Herndon-Dudley syndrome patients.SIGNIFICANCE STATEMENT Thyroid hormones (THs) are essential to establish the stereotypical layered structure of the human forebrain during embryonic development. Before their action on gene expression, THs require cellular uptake, a process facilitated by the TH transporter monocarboxylate transporter 8 (MCT8). We investigated how and when dysfunctional MCT8 can induce brain lesions associated with the Allan-Herndon-Dudley syndrome, characterized by psychomotor retardation. We used the layered chicken optic tectum to model cortical development, and induced MCT8 deficiency in neural progenitors. Impaired cell proliferation, migration, and differentiation resulted in an underdeveloped optic tectum and a severe reduction in nerve cells. Our data underline the need for MCT8-dependent TH uptake in neural progenitors and stress the importance of local TH action in early development.status: publishe

Decreased thyroid hormone signaling accelerates the reinnervation of the optic tectum following optic nerve crush in adult zebrafish

The regenerative capacity of the adult mammalian central nervous system (CNS) is poor and finding ways to stimulate long distance axonal regeneration in humans remains a challenge for neuroscientists. Thyroid hormones, well known for their key function in CNS development and maturation, more recently also emerged as molecules influencing regeneration. While several studies investigated their influence on peripheral nerve regeneration, in vivo studies on their role in adult CNS regeneration remain scarce. We therefore investigated the effect of lowering T3 signaling on the regeneration of the optic nerve (ON) following crush in zebrafish, a species where full recovery occurs spontaneously. Adult zebrafish were exposed to iopanoic acid (IOP), which lowered intracellular 3,5,3'-triiodothyronine (T3) availability, or to the thyroid hormone receptor β antagonist methylsulfonylnitrobenzoate (C1). Both treatments accelerated optic tectum (OT) reinnervation. At 7days post injury (7dpi) there was a clear increase in the biocytin labeled area in the OT following anterograde tracing as well as an increased immunostaining of Gap43, a protein expressed in outgrowing axons. This effect was attenuated by T3 supplementation to IOP-treated fish. ON crush induced very limited cell death and proliferation at the level of the retina in control, IOP- and C1-treated fish. The treatments also had no effect on the mRNA upregulation of the regeneration markers gap43, tub1a, and socs3b at the level of the retina at 4 and 7dpi. We did, however, find a correlation between the accelerated OT reinnervation and a more rapid resolution of microglia/macrophages in the ON and the OT of IOP-treated fish. Taken together these data indicate that lowering T3 signaling accelerates OT reinnervation following ON crush in zebrafish and that this is accompanied by a more rapid resolution of the inflammatory response.publisher: Elsevier articletitle: Decreased thyroid hormone signaling accelerates the reinnervation of the optic tectum following optic nerve crush in adult zebrafish journaltitle: Molecular and Cellular Neuroscience articlelink: http://dx.doi.org/10.1016/j.mcn.2015.04.002 content_type: article copyright: Copyright © 2015 Elsevier Inc. All rights reserved.status: publishe

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

Mosaic Expression of Thyroid Hormone Regulatory Genes Defines Cell Type-Specific Dependency in the Developing Chicken Cerebellum

The cerebellum is a morphologically unique brain structure that requires thyroid hormones (THs) for the correct coordination of key cellular events driving its development. Unravelling the interplay between the multiple factors that can regulate intracellular TH levels is a key step to understanding their role in the regulation of these cellular processes. We therefore investigated the regional/cell-specific expression pattern of TH transporters and deiodinases in the cerebellum using the chicken embryo as a model. In situ hybridisation revealed expression of the TH transporters monocarboxylate transporter 8 (MCT8) and 10 (MCT10), L-type amino acid transporter 1 (LAT1) and organic anion transporting polypeptide 1C1 (OATP1C1) as well as the inactivating type 3 deiodinase (D3) in the fourth ventricle choroid plexus, suggesting a possible contribution of the resulting proteins to TH exchange and subsequent inactivation of excess hormone at the blood-cerebrospinal fluid barrier. Exclusive expression of LAT1 and the activating type 2 deiodinase (D2) mRNA was found at the level of the blood-brain barrier, suggesting a concerted function for LAT1 and D2 in the direct access of active T3 to the developing cerebellum via the capillary endothelial cells. The presence of MCT8 mRNA in Purkinje cells and cerebellar nuclei during the first 2 weeks of embryonic development points to a potential role of this transporter in the uptake of T3 in central neurons. At later stages, together with MCT10, detection of MCT8 signal in close association with the Purkinje cell dendritic tree suggests a role of both transporters in TH signalling during Purkinje cell synaptogenesis. MCT10 was also expressed in late-born cells in the rhombic lip lineage with a clear hybridisation signal in the outer external granular layer, indicating a potential role for MCT10 in the proliferation of granule cell precursors. By contrast, expression of D3 in the first-born rhombic lip-derived population may serve as a buffering mechanism against high T3 levels during early embryonic development, a hypothesis supported by the pattern of expression of a fluorescent TH reporter in this lineage. Overall, this study builds a picture of the TH dependency in multiple cerebellar cell types starting from early embryonic development.status: publishe