Defective thyroid hormone transport to the brain leads to astroglial alterations

Defective thyroid hormone transport to the brain leads to astroglial alterations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Defective thyroid hormone transport to the brain leads to astroglial alterations Marina Guillén-Yunta, Ángel García-Aldea, Víctor Valcárcel-Hernández, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3825537/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : Allan-Herndon-Dudley syndrome (AHDS) is a rare X-linked disorder that causes severe neurological damage, for which there is no effective treatment. AHDS is due to inactivating mutations in the thyroid hormone transporter MCT8 that impair the entry of thyroid hormones into the brain, resulting in cerebral hypothyroidism. However, the pathophysiology of AHDS is still not fully understood and this is essential to develop therapeutic strategies. Based on evidence suggesting that thyroid hormone deficit leads to alterations in astroglial cells, including gliosis, in this work we have evaluated astroglial impairments in MCT8 deficiency. Methods : We conducted magnetic resonance imaging on both control subjects and MCT8-deficient patients to examine changes in brain cytoarchitecture. Moreover, to gain further understanding on these alterations in brain cytoarchitecture and the astroglial population, we have performed histological and immunohistochemical approaches in autopsy brain samples from an 11-year-old and a 30 th gestational week MCT8-deficient subjects in comparison to brain samples from control subjects at similar ages. Findings from MCT8-deficient subjects were validated and further explored in a mouse model of the AHDS. Results : Magnetic resonance imaging showed changes indicative of alterations in brain cytoarchitecture in MCT8-deficient patients. Further studies confirmed changes in the astroglial population in MCT8 deficiency that arise early in brain development and persist at adult stages, revealing an abnormal distribution, density, and morphology of cortical astrocytes, compatible with an astrogliosis-like phenotype at adult stages. Conclusions : We have identified astrocytes as potential novel therapeutic targets in AHDS. In addition, we propose ADC imaging as a tool to monitor the progression of neurological impairments and potential effects of treatments in MCT8 deficiency. Thyroid hormones Thyroid hormone transporters MCT8 Astroglia MCT8 deficiency MRI. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 BACKGROUND Allan-Herndon-Dudley Syndrome (AHDS) or MCT8 deficiency is a rare X-linked disorder caused by inactivating mutations in the gene that codifies for the monocarboxylate transporter 8 (MCT8) ( 1 – 3 ). MCT8 is a transmembrane transporter highly specific for thyroid hormones (THs), both thyroxine (T4) and the transcriptionally active form 3,5,3’-triiodothyronine (T3) ( 4 ). This syndrome is characterized by peripheral hyperthyroidism concomitant with brain hypothyroidism. Patients also present severe neurological damage, psychomotor retardation, and global developmental delay. Moreover, the majority of affected boys have profound intellectual disability (IQ < 30), speech difficulties, as well as central hypotonia, spastic paraplegia, and dystonic movements ( 5 – 7 ). The neurological impairments have been linked to impaired transport of THs across the brain barriers ( 8 ), where MCT8 is highly expressed in humans ( 9 , 10 ), leading to low T3 and T4 content in the brain ( 11 ). However, despite these insights, the neuropathophysiological mechanisms underlying MCT8 deficiency are not well understood and there is no effective treatment to palliate the severe neurological impairments in MCT8-deficient patients. In order to develop therapeutic approaches to improve the neurological alterations in MCT8-deficient patients, it is essential to have a complete understanding of the nature of these alterations. The defects identified to date in the brain of MCT8-deficient patients are limited and point to a wide range of alterations including deficient myelination (reviewed in ( 12 )), decreased myelinated axonal diameter ( 13 ), altered cortical and cerebellar structure, defects at the synapse with deficient expression of synaptophysin, defects at GABAergic interneurons with abnormal parvalbumin and calbindin-D28k expression ( 11 ), as well as neurovascular unit disruption ( 14 ). The complex neurological phenotype in MCT8-deficient patients seems to be arising from several brain impairments. The identification of additional pathological mechanisms mediating the brain alterations of patients will provide valuable information to design appropriate treatments. THs have been shown to regulate the maturation and function of astrocytes both in vitro and in vivo ( 15 – 17 ). Astroglial cells express TH receptors (TRs) ( 18 ), indicating that they are direct targets of THs action ( 17 ). Indeed, astrocytes express the MCT8 transporter in human ( 9 , 19 ), monkey ( 19 ), and mouse ( 17 , 20 ). Astrocytes also express the enzyme deiodinase 2 (DIO2) which locally generates the main cerebral pool of T3 from T4 ( 21 ). There is recent evidence suggesting that brain hypothyroidism leads to astrogliosis ( 22 – 24 ). Astrogliosis is a reaction of astrocytes to disturbed homeostasis in the brain, with changes in the number of astrocytes, their morphology, and function. While changes in astrocytes might initiate repair processes in the brain, they can also be detrimental by leading to secondary damage such as neuronal death or abnormal neuronal activity ( 25 – 27 ). In view of this, we hypothesized that MCT8 deficiency affects the astroglial population which may be one of the underlying causes for some of the neurological defects in MCT8-deficient patients. To test this hypothesis, we analyzed apparent diffusion coefficient (ADC) imaging values from MCT8-deficient patients and we identified histopathological abnormalities associated with altered cytoarchitecture, including gliosis ( 28 , 29 ). Alterations in astroglia were confirmed by immunolabeling brain samples of an 11-year-old and a 30th gestational week MCT8-deficient subjects with antibodies against astroglial markers. These findings were validated and further explored in a mouse model of AHDS. Our findings confirm changes in the astroglial population in MCT8 deficiency that arise early in brain development and persist at adult stages, revealing an abnormal distribution, density, and morphology of cortical astrocytes, compatible with an astrogliosis-like phenotype at adult stages. MATERIALS AND METHODS Study design This study examined brain cytoarchitecture alterations, in particular in the astroglia population, as a pathophysiological mechanism mediating the severe brain impairments in MCT8 deficiency. First, we calculated the ADC imaging values obtained from magnetic resonance imaging in the cerebral cortex and the striatum of 11 MCT8-deficient patients and 11 control subjects in a single-blinded fashion. Next, we further studied potential brain alterations by histochemistry and immunohistochemistry in autopsy brain samples from an 11-year-old and a 30th gestational week MCT8-deficient subjects in comparison to brain samples from control subjects at similar ages. In addition, we studied brain samples from a previously validated mouse model of the AHDS ( Mct8/Dio2 KO, ( 30 )). Data from control and Mct8/Dio2 KO mice were acquired in a single-blinded manner with a number assigned to each animal unrelated to their genotype. Sample size for immunohistochemistry (n = 4) was selected according to previous experience ( 13 , 30 , 31 ). Magnetic resonance imaging acquisition and processing Brain MR Imaging (MRI) acquisition was performed in a cohort of 22 boys between 3 and 13 years old including 11 MCT8-deficient patients and 11 age-matched controls (see Table 1 ) using a 3.0T scanner (Magnetom Skyra-Siemens Heathinners, Erlangen, Germany) at the Medical University of South Carolina. The examination included T1-weighted images acquired with an Inversion Recovery sequence (TE = 2.5 ms, TR = 1900 ms, TI = 900 ms, matrix 256x256, voxel-size = 0.86x0.86x0.86 mm³) and diffusion-weighted imaging (DWI) with two b-values: b = 0 and b = 1000 s/mm² to estimate ADC maps (TR = 6400ms, TE = 98ms, voxel-size = 1.2x1.2x5mm³). DWI images were processed to obtain the ADC maps. First, elastic registration to T1-weighted images was performed to remove EPI distortion using ANTs ( 32 ), and ADC was estimated from the images acquired with b = 0 and b = 1000 s/mm². T1-weigthed images were processed with Freesurfer 7.1.1 software ( 33 ) to obtain brain parcellation into anatomical regions. This parcellation was then translated to the ADC map, and average ADC value in the regions of interest was obtained. Based in the psychomotor impairments and profound intellectual disability of the patients, the regions of interest were the cerebral cortex (left and right) and striatum (including caudate and putamen regions identified by Freesurfer). Table 1 Features of the subjects enrolled in Magnetic Resonance Imaging (MRI) studies. Subject Diagnosis Sex Age (y) 1 Control M 9 2 Control M 13 3 Control M 3 4 Control M 6 5 Control M 7 6 Control M 3 7 Control M 5 8 Control M 3 9 Control M 4 10 Control M 10 11 Control M 5 12 AHDS M 9 13 AHDS M 13 14 AHDS M 3 15 AHDS M 7 16 AHDS M 6 17 AHDS M 3 18 AHDS M 5 19 AHDS M 3 20 AHDS M 3 21 AHDS M 10 22 AHDS M 4 Human tissue samples As in López-Espíndola et al. ( 11 ), we used postmortem brain paraffin samples from an 11-year-old subject with a mutation (Q96X) in the SLC16A2 gene, with severe psychomotor impairment, high serum T3, low rT3 and T4, and mildly elevated TSH. Previously reported MRI showed a mildly delayed myelination at 9 months, along with a slight dilatation of the lateral ventricles’ frontal horns at 6.5 years. The cause of death was a respiratory failure secondary to aspiration pneumonia. The paraffin blocks were provided by the Sydney Children’s and Prince of Wales Hospitals, Randwick, Australia. Control samples came from a 10-year-old girl and a 12-year-old boy whose causes of death were acute pulmonary edema during a surgical procedure and diffuse lymphocytic myocarditis, respectively. Paraffin blocks were kindly provided by the IdiPAZ Biobank (PT20/00004), and the Biobanc per a la Investigació de l’Hospital Infantil Sant Joan de Déu, Barcelona (integrated into the Spanish National Biobanks Network) and they were processed following standard operating procedures with the appropriate approval of the Ethics and Scientific Committees. As in López-Espíndola et al. ( 11 ), we also used a 30th gestational week male fetus with a mutation (L494P) in the SLC16A2 gene identified by amniocentesis. Pregnancy was terminated upon parental request and approval by an officially designated committee (Wolfson Medical Center, Holon, Israel, and the Sackler School of Medicine, Tel Aviv, Israel) and paraffin blocks were provided by the Wolfson Medical Center. As a control, a 30th gestational week male fetus aborted due to placental abruption was used. Paraffin blocks were provided by the IdiPAZ Biobank (PT20/00004). All paraffin blocks were sliced in a microtome (Microm, HM 310) at 7 µm of thickness for immunohistochemistry assays. Experimental animals Animals were housed in temperature- and light-controlled conditions at 22 ± 2°C on a 12:12 light-dark cycle (lights on at 7 AM), with access to food and water ad libitum . Since AHDS is an X-linked disease affecting almost exclusively males, only wild-type (WT) and Mct8 −/y /Dio2 −/− ( Mct8/Dio2 KO) male mice were used in all studies. Animals were euthanized at postnatal day 7 (P7), P15, 3, and 6 months of age for immunohistochemical analysis. Mct8 KO mice were originally produced by Dumitrescu et al. 2006 ( 34 ) and Dio2 KO by Schneider et al. 2001 ( 35 ). Mct8/Dio2 KO mice were bred at the animal facility of the Instituto de Investigaciones Biomédicas Sols-Morreale by crossing Mct8 +/y /Dio2 −/− males and Mct8 −/+ /Dio2 −/− females. Animals were housed in groups of up to 6 mice per cage with environmental enrichment consisting of nesting material and a cardboard tube. All genotypes were confirmed by PCR of ear DNA as described ( 30 ). For histological procedures, mice were anesthetized with ketamine (75 µg/g of body weight) and medetomidine hydrochloride (1 µg/g of body weight) and transcardially perfused with 4% paraformaldehyde in 0.1M phosphate buffer (PB). Brains were removed, post-fixed overnight in 4% paraformaldehyde in 0.1M PB, cryoprotected in 30% sucrose, and cut into 25 µm coronal sections on a cryostat. All sample collections were performed between 09.00 and 12.00 a.m. with time-matched experimental controls run in parallel. Immunohistochemistry and histology Human brain samples : Immunohistochemical procedures for human brain samples were performed as previously described ( 11 , 13 ). Tissue sections were deparaffinized and re-hydrated. To facilitate the antigen-antibody reaction, sections were incubated for 20 min at 95 °C with the Envision FLEX Low pH solution for antigen retrieval (DAKO, K8005). Endogenous peroxidase was blocked by using 3% hydrogen peroxide in distilled water for 15 min. Samples were blocked with phosphate-buffered saline (PBS) containing 4% bovine serum albumin (BSA, Sigma A4503), 0.1% Triton X-100, 0.1 M Lysine, and 5% normal goat serum (Vector Laboratories, S-1000) for 1 h. Samples were incubated overnight at 4 °C with the anti-Glial Fibrillary Acidic Protein (GFAP) antibody (1:200, DAKO Z0334) in PBS containing 4% BSA, 0.1% Triton X-100, and 1% normal goat serum. Tissues were washed in PBS and incubated for 1 h at RT with the corresponding biotinylated secondary antibody (Vector Laboratories) in PBS containing 4% BSA, 0.1% Triton, and 1% serum. In order to amplify the immune signal, tissue samples were incubated for 1 h in the dark with Avidin-Biotin-Complex (ABC Elite Kit; Vector Laboratories, #32050), according to the manufacturer’s instructions. Finally, tissue sections were incubated with diaminobenzidine (0.5 mg/mL, Sigma, D5637) in 0.01% hydrogen peroxide. Preparations from the 30th gestational week male fetuses were counterstained with Harris hematoxylin for a few seconds (Sigma, HHS32). The sections were dehydrated, cleared in xylene, and cover-slipped with DePeX (Serva, 18243). Negative controls omitting the primary antibody run in parallel displayed no immunopositive signal. Mouse brain samples : The immunodetection was performed in free-floating sections as previously described ( 13 , 30 , 31 ). For Aldehyde Dehydrogenase 1 Family Member L1 (ALDH1L1) immunolabelling, sections were incubated in Envision FLEX Low pH solution for antigen retrieval buffer (DAKO, K8005) for 12 min at 95°C. This step was omitted for GFAP immunostaining. After several washes in PBS, the endogenous peroxidase activity was blocked using 10% methanol and 3% hydrogen peroxide in PBS for 15 min in the dark. Immunolabeling using anti-GFAP (1:3000, DAKO Z0334) and ALDH1L1 (1:1000, Proteintech 17390-1-AP) was performed as described for human samples. Genotypes and time-matched experimental controls were run in parallel to avoid methodological differences. Negative controls omitting the primary antibody, also run in parallel, displayed no immunopositive signal. Image analysis and quantification Immunohistochemically-labeled brain sections were examined under brightfield illumination using a Nikon Eclipse 80i (Nikon Corp., Tokyo, Japan) microscope and microphotographs were taken with a Nikon DSFi1 digital camera. Cortical lamination was determined by analyzing parallel tissue sections stained with hematoxylin-eosin in human and Nissl staining in mouse. Quantification of GFAP immunopositive (GFAP+) cell density in the mouse motor cortex was evaluated by counting all the GFAP + cells in the secondary motor cortex (M2) in four sections between bregma 1.18 to -0.58 in four different animals per genotype. M2 cortex (layers I to VI) was delineated using a 20x (numerical aperture 0.5) and GFAP + cells were counted using a 40x objective (numerical aperture 0.75). Statistics Data were expressed in box and whisker plots. Statistical analyses were performed using GraphPad Prism Software ( www.graphpad.com ). Normality of the data was assessed by the Shapiro–Wilk test. Means between two groups were compared with a 2-tailed unpaired Student’s t-test for parametric data and with a 2-tailed Mann Whitney test for non-parametric data. Significant differences were represented as * p < 0.05; ** p < 0.01, and *** p < 0.001. In human MRI studies, the effect of age and condition on ADC values was estimated using a General Linear Model with one fixed factor (Control, MCT8-deficient) and age as a covariate. Pearson’s correlation coefficient was used to explore the statistical relationship between the cerebral cortex and the striatum. Data are presented in box-plots representing the 25th (bottom), 50th (middle-line) and 75th (top) quartiles with whiskers extending from minimum to maximum values, otherwise specified. RESULTS Brain water diffusivity is altered in MCT8-deficient patients The brains from 11 MCT8-deficient patients and 11 age-matched controls ranging from 3 to 13 years old were non-invasively studied using brain DWI. DWI allows the assessment of the water ADC, a measurement of tissue water diffusivity which is altered in different pathological conditions. Changes in the ADC value have been associated with alterations in brain cytoarchitecture, including gliosis ( 28 , 29 ). The analysis of ADC was focused on the cerebral cortex (Fig. 1 a), a brain region involved in high-order cognitive processing, and the striatum, a brain structure associated with movement disorders (Fig. 1 b). Figure 1 c and d depict each individual ADC value versus the age of the corresponding patient in the cerebral cortex and striatum, respectively. We observed that the ADC value did not change with age in either MCT8-deficient or control subjects in the cerebral cortex and the striatum. Most importantly, we observed a significant increase in ADC values in MCT8-deficient patients compared to controls in both the cerebral cortex (F( 1 , 18 ) = 29.339, p < 0.0001, Fig. 1 e) and the striatum F( 1 , 19 ) = 33.665, p < 0.0001, Fig. 1 f). MCT8 deficiency leads to persistent astroglial alterations in the cerebral cortex To assess whether the increased ADC values in MCT8-deficient patients were associated to astroglial alterations, we evaluated the density and morphology of astroglial populations in brain samples of an 11-year-old MCT8-deficient patient. This was performed by immunohistochemistry against the astroglial marker GFAP, a major component of intermediate filaments in astrocytes, in the different brain regions available which included the motor, sensory, and frontal cortices as well as the striatum in the basal ganglia. In the motor cortex of the control sample, GFAP immunoreactivity in the grey matter was mainly found in cell bodies and processes of subpial astrocytes forming the glia limitans and in interlaminar astrocytes of the cortical layer I (Fig. 2 a,c). Subpial astrocytes showed intense GFAP labeling, and the cell bodies and proximal radial processes of interlaminar astrocytes showed medium-intensity GFAP staining (Fig. 2 c). There were scarce GFAP + astrocytes with radial processes characteristic of protoplasmic astrocytes across the rest of the layers of the cortex, usually associated to microvessels (Fig. 2 e,g,i). Fibrous astrocytes were found in the subcortical white matter (WM), with small cell bodies and processes (Fig. 2 k). The intensity of GFAP expression in the subcortical WM was medium to low and similar between cell bodies and processes. The motor cortex of the MCT8-deficient subject showed a different distribution of GFAP + cells in comparison to the control subject. GFAP + staining in the glia limitans displayed less density of subpial astrocytes than in control samples, but a similar density of interlaminar astrocytes in layer I (Fig. 2 b,d). In contrast to the control, GFAP + cells with astrocytic features similar to the ones present in layer I were present throughout all cortical layers (Fig. 2 f,h,j), particularly at the gyrus, and were less pronounced at the banks and the sulcus where stained astrocytes were almost exclusively associated to microvessels at layers II-VI. The MCT8-deficient subcortical WM presented a higher density of fibrous astrocytes in comparison to controls, with intensely stained cell bodies and shorter processes (Fig. 2 l). In conclusion, the MCT8-deficient subject presented fewer subpial astrocytes at the glial limitans, and an increased number of fibrous astrocytes across layers II-VI and the subcortical WM, where astrocyte morphology also differed from control samples. Similar findings were observed at the sensory and frontal cortices ( Supplemental Fig. 1, online Resource 1 ). Control sensory and frontal cortices showed subpial astrocytes with intense GFAP expression, interlaminar astrocytes in layer I with medium GFAP intensity staining in cell bodies and proximal radial processes; scarce GFAP + astrocytes across the rest of cortical layers; and low GFAP stained cell bodies and processes in the subcortical WM. As observed in the motor cortex, GFAP + cells in the MCT8-deficient sensory and frontal cortices were present throughout all cortical layers, mainly in the gyral convexity, and the density of fibrous astrocytes in the subcortical WM was higher than in control samples. The analysis of the GFAP + astrocytes in the basal ganglia was performed in the available tissue, which included the caudate and putamen of the striatum. Prominent GFAP immunolabeling was identified in the proximity of blood vessels both in the control (Fig. 3 a) and MCT8-deficient (Fig. 3 b) samples. Even though there were no apparent differences in the density of GFAP + cells, the staining intensity of GFAP + cells appeared to be higher in MCT8-deficient samples in comparison to the control subject (Fig. 3 c,d). In order to validate and further explore the astroglial alterations found in the human MCT8-deficient samples, we performed immunohistochemistry against two different astroglial markers, GFAP and ALDH1L1, in brain samples of a validated model of the AHDS: the Mct8/Dio2 KO mouse model ( 30 ). Studies were initially performed in 3-month-old mice, an age that corresponds to an 11-year-old human ( 36 ), focusing on the motor cortex and the basal ganglia, as for the human samples. In WT animals, GFAP + astrocytes were present mainly in the upper layers of the motor cortex, with restricted GFAP signal to the soma and initial part of the astrocytic processes (Fig. 4 a,c,e). In contrast, in the motor cortex of Mct8/Dio2 KO mice astroglial cells were found throughout all cortical layers showing an increased cell density with higher GFAP signal in astroglial processes and cell bodies than in WT animals (2.41 fold-change increase; t( 6 ) = 25.11, p < 0.0001, Fig. 4 b,d,f,m). ALDH1L1 immunolabeling exhibited a broader expression pattern in WT samples in comparison to GFAP in the motor cortex (Fig. 4 g,I,k), as previously described ( 37 ). In the motor cortex, ALDH1L1 immunostaining was present in all cortical layers of WT and Mct8/Dio2 KO mice, although the ALDH1L1 + cell density was higher in Mct8/Dio2 KO samples (1.46 fold-change increase; t( 6 ) = 9.085, p < 0.0001, Fig. 4 h,j,l,n). Notably, ALDH1L1 staining intensity in Mct8/Dio2 KO mice was higher in astroglial processes and cell bodies than in WT animals (Fig. 4 i-l). In the striatum of the basal ganglia, GFAP and ALDH1L1 immunolabeling displayed a similar density of GFAP+ (Fig. 5 a,b) and ALDH1L1+ (Fig. 5 e,f) cells in the WT and Mct8/Dio2 KO mice, resembling the findings in human samples. Moreover, GFAP staining intensity appeared to be higher in Mct8/Dio2 KO than in WT mice (Fig. 5 c,d), and no apparent differences in staining intensity were observed for ALDH1L1 immunolabeling. To discern the relative roles of DIO2 and MCT8 proteins in the immunostaining pattern of GFAP, Dio2 KO, and Mct8 KO animals were also analyzed in the motor cortex. This analysis confirmed the increase in the number of GFAP + astrocytes in Mct8/Dio2 KO mice in comparison to WT (2.41 fold-change increase; Tukey's multiple comparisons test, p < 0.0001). Notably, there were no differences in the number of GFAP + astrocytes between WT and Dio2 KO mice (Tukey's multiple comparisons test, p = 0.0835) and a small increase in the number of GFAP + astrocytes in Mct8 KO mice in comparison to WT (1.25 fold-change increase; Tukey's multiple comparisons test, p = 0.0101, Supplementary Fig. 2, online Resource 1 ). To assess if these changes in the morphology and number of astroglial cells were persistent at later stages, immunolabeling against GFAP and ALDH1L1 was performed in 6-month-old WT and Mct8/Dio2 KO mice. The outcomes resembled the findings in 3-month-old mice: there was an increased number of GFAP+ (3.21 fold-change increase; t( 6 ) = 11.57, p < 0.0001) and ALDH1L1+ (1.47 fold-change increase; t( 6 ) = 8.88, p < 0.0001) astrocytes throughout all the cortical layers with increased GFAP and ALDH1L1 labeling in the soma and processes of the astrocytes in the motor cortex of Mct8/Dio2 KO mice compared to WT ( Supplementary Fig. 3, online Resource 1 ). The increase in GFAP + cells across all cortical layers are indicative of persistent astroglial alterations, compatible with an astrogliosis-like phenotype, in the cerebral cortex of a mouse model of AHDS. MCT8 deficiency leads to astroglial developmental alterations To assess whether astroglial alterations are present during early brain development or whether they are acquired at later developmental stages, we performed immunostaining against GFAP in the occipital cortex from a 30th gestational week MCT8-deficient fetus. The occipital cortex of the MCT8 deficient fetus showed poorly defined lamination, smaller thickness of cortical layers (in particular layers II, III, and IV), and higher cell density in comparison to a control subject (Fig. 6 a,b), as previously described ( 11 ). In layer I of the occipital cortex, GFAP immunostaining was altered in the MCT8-deficient fetus with decreased, or even absent, GFAP + signal at cell bodies and processes (Fig. 6 d). Layers II-IV did not present distinctive GFAP labeling either in the control or the MCT8-deficient patient (Fig. 6 e-h). Layers V and VI presented GFAP + radial glia processes both in the control and the MCT8-deficient fetus (Fig. 6 i,j). Striking differences were observed in the transition between layer VI and the subcortical WM and in subcortical WM itself, where the control sample displayed well-organized radial glial fibers with highly polarized astrocytes, while the MCT8-deficient fetus presented less GFAP + radial glia fibers and less polarized processes emerging from GFAP + astrocytes (Fig. 6 k,l). These findings suggest that the astrogliosis-like phenotype observed in the 11-year-old MCT8-deficient subject is not present during fetal brain development, however, there seem to be defects on radial glia fibers. In order to validate and further explore the astroglial alterations found in human MCT8-deficient samples during brain development, we performed immunohistochemistry against GFAP in Mct8/Dio2 KO mouse occipital cortices, in particular, in the visual cortex. We studied mice at postnatal day 7 (P7), the stage of early differentiation of astrocytes ( 38 , 39 ), and at P15, which corresponds to the period of late differentiation and maturation of astrocytes, equivalent to a 30th gestational week human fetus ( 38 , 39 ). Similar to the findings observed in the MCT8-deficient human fetal occipital cortex, Nissl staining of the visual cortex revealed that the cytoarchitecture of the cortical layers is disorganized in Mct8/Dio2 KO animals in comparison to WT, both at P7 and P15. At P7, the thickness of the cortical layers (in particular layers II-IV) of Mct8/Dio2 KO mice was decreased in comparison to WT mice (Fig. 7 a,b). Mct8/Dio2 KO also displayed higher cell density in the upper layers, lower cell density in the infragranular layers, and poorly defined boundaries between cortical layers compared to WT mice (Fig. 7 a,b). At P15, Mct8/Dio2 KO animals presented poorly defined boundaries between cortical layers in comparison to WT mice, however, there were no apparent differences in the cell density between the two genotypes (Fig. 7 c,d). Analysis of the distribution of astrocytes in the different layers of the visual cortex of WT and Mct8/Dio2 KO animals at P7 showed a very similar distribution of GFAP + cells in both genotypes, with immunopositive cells located mainly in layer I (Fig. 7 e,f). However, Mct8/Dio2 KO mice presented a decrease in immunolabeled GFAP + fibers, most likely radial glia processes, in comparison to WT (Fig. 7 e-h). At P15, radial glia processes were already absent in both WT and Mct8/Dio2 KO animals (Fig. 7 i-l). At this age, GFAP + astrocytes in WT animals concentrated mainly in layers I, II, and VI (Fig. 7 i,k), while in Mct8/Dio2 KO animals there was an increase in GFAP + astrocytes distributed throughout all cortical layers (Fig. 7 j,l). In addition, GFAP + astrocytes presented greater intensity of GFAP labeling, as well as an increased number of processes and ramifications in Mct8/Dio2 KO mice in comparison to WT (Fig. 7 i-l). These findings validate the observations in the human samples indicating that the astrogliosis-like phenotype observed in adult Mct8/Dio2 KO mice is not present during early brain development, and that it develops between P7 and P15. Additionally, findings in Mct8/Dio2 KO mice support defects in the radial glia. DISCUSSION Given the importance of THs action during astrocyte development, as well as recent evidence suggesting that hypothyroidism might lead to gliosis ( 22 – 24 ), in this work we have evaluated possible alterations in the astroglial population including a potential state of astrogliosis as an additional pathological mechanism mediating the severe neurological impairments in MCT8-deficient patients. We have analyzed MR images to identify in vivo alterations in the brain cytoarchitecture of MCT8-deficient patients, which may be a predictor of gliosis and tissular damage. Analysis of brain water diffusivity by ADC, frequently seen with gliosis in different cerebral pathologies such as adrenoleukodystrophies or Alzheimer’s disease ( 29 , 40 ), revealed increases in ADC values in the cerebral cortex and the striatum of MCT8-deficient patients in comparison to controls, indicating brain microstructure alterations. Increases in ADC values might also be associated with potential gliosis in MCT8 deficiency. Indeed, we have identified important alterations in the number and distribution of astroglial cell populations in the motor, sensory, and frontal cortices from an 11-year-old human MCT8-deficient subject. These included increased number of astrocytes particularly across layers III-VI and in the subcortical WM, suggestive of reactive astrogliosis in MCT8 deficiency. However, no apparent changes in the number of astrocytes were observed in the basal ganglia of the MCT8-deficient subject or in Mct8/Dio2 KO mice. Increases in ADC values in the striatum of MCT8-deficient patients, in combination with previously identified changes in fractional anisotropy by MRI in Mct8/Dio2 KO mice [13], may be indicative of axonal degeneration in the striatum [28]. This observation was validated in a mouse model of MCT8 deficiency at 3 months of age, the correlating age to an 11-year-old human ( 36 ). We found an increased density of GFAP + and ALDH1L1 + cells in the cerebral cortex of Mct8/Dio2 KO mice in comparison to control samples, confirming the existence of astroglial alterations, compatible with astrogliosis, associated with MCT8 deficiency at preadolescent stages. In addition, the presence of alterations in GFAP + immunostaining in Mct8/Dio2 KO mice at 6 months of age is suggestive of persistent astroglial alterations in MCT8 deficiency at later stages. Moreover, the mild alterations in GFAP immunostaining in single Mct8 KO mice indicate that astroglial alterations in MCT8 deficiency are likely due to the state of brain hypothyroidism ( 11 , 30 ) arising from impaired THs transport into the brain across the brain barriers ( 8 , 41 , 42 ), rather than to the lack of MCT8 in the astrocytes. In the absence of MCT8, THs transport into astrocytes could be compensated by the presence of additional THs transporters, such as LAT1 ( 20 ). Indeed, there is increasing evidence indicating that brain hypothyroidism leads to astrogliosis. It has been observed that hypothyroidism induced by anti-thyroid drugs increases the number of GFAP + cells in the cortex ( 22 ) and hippocampus ( 22 , 24 ) and that brain-hypothyroid-mice deficient in MCT8 and the organic anion transporting protein OATP1C1 present an elevated number of astrocytes in the corpus callosum ( 23 ). Our findings further support this emerging body of literature linking hypothyroidism to astrogliosis and extend these observations to humans. Such revelations could have an important impact beyond the MCT8 deficiency field in other hypothyroid conditions such as, for example, those derived from congenital hypothyroidism, Hashimoto's disease, thyroiditis, low iodine diet, or endocrine disruptors. Further studies are necessary to determine if the astroglial alterations observed in the cerebral cortex are a result of the brain insults derived from MCT8 deficiency, due to alterations in the development of the astroglial lineage, or both. Here we have observed that brain samples from a 30th gestational week MCT8-deficient fetus presented less GFAP + cells only in layer I of the occipital cortex and no changes in the number of astrocytes were observed in the visual cortex of Mct8/Dio2 KO mice at P7. However, by P15 there was an evident increase in the number of astrocytes in the cortex of Mct8/Dio2 KO mice. On one hand, this could suggest that the increase in GFAP + cells is a response to early brain insults that take place between P7 and P15. On the other hand, this could point to alterations arising during the generation of astrocytes. One of the main sources of astrocytes are neural stem cells (NSCs) that, following the “gliogenic switch”, stop generating neurons and predominantly generate oligodendrocyte and astrocyte lineage cells ( 38 , 39 ). The differentiation of NSCs into specific glial populations is determined by the sustained expression of a single basic helix-loop-helix transcription factor, in particular, Olig2 expression will give rise to oligodendrocyte precursor cells (OPCs) and Hes1 to astrocytes ( 43 ). Interestingly, Olig2 is a positively T3-regulated gene ( 44 ) and T3 has been shown to negatively regulate other basic helix-loop-helix transcription factors from the Hes gene family such as Hes7 and Hes5 ( 17 , 44 ). This suggests that the brain hypothyroid state in MCT8 deficiency might be promoting the generation of astrocytes from NSCs which could be the underlying cause for the elevated number of astrocytes associated with MCT8 deficiency. Regardless of the nature of the increase in the number of astrocytes at P15 in Mct8/Dio2 KO mice, these alterations in the astroglial population could be severely impacting other neurodevelopmental processes. For example, synaptic pruning, a synaptic maturation process with a synapse elimination phase, is primarily mediated by astroglial cells ( 45 ). This illustrates how alterations in astroglia could lead to other defects, therefore affecting the refinement of neuronal circuits during development. Finally, we have also observed less GFAP + radial glia processes in the MCT8-deficient fetus in comparison to controls. This finding was corroborated in Mct8/Dio2 KO animals at P7 that also presented fewer radial glia processes than WT in the visual cortex. This is of relevance as we have previously identified the radial glia as an important regulator of THs availability to the human developing brain, as it presents the TH transporters MCT8 and OATP1C1 as well as DIO2 and the enzyme deiodinase 3 (DIO3) ( 9 ). This suggests that impaired radial glia development in MCT8 deficiency may impact the local availability of THs to neural cells that are dependent on this THs availability pathway. The alterations in radial glia processes here identified are likely mediating the cytoarchitecture alterations observed both in the MCT8-deficient human fetus and Mct8/Dio2 KO animals at P7 and P15, which include higher cell density, decreased thickness of cortical layers, and poorly defined lamination. This could be because, during corticogenesis, newly generated neuroblasts use radial glia fibers as a scaffold for migration towards the upper cortical layers ( 46 ). Nevertheless, the observed reduction in cortical thickness could also be due to defects in proliferation and a decreased number of neural progenitors in hypothyroid conditions, as previously described ( 47 ). CONCLUSIONS Based on the current findings we conclude that MCT8 deficiency leads to alterations in astroglia, consistent with astrogliosis, from early stages of development that are persistent at adult stages. Impaired T3 signaling in NSCs could be a contributing factor to the elevated astrocyte number and changes in astrocyte morphology. Defects in radial glia might be underlying the alterations in brain cytoarchitecture identified by ADC and histochemistry in MCT8-deficient patients. Moreover, our findings point to ADC analysis as a possible in vivo non-invasive marker of cytoarchitecture alterations in MCT8-deficiency and might be a predictor of astroglia imbalance to evaluate the progression of neurological impairments in patients, as well as the outcome of potential therapeutic strategies. In summary, MCT8 deficiency leads to an astrogliosis-like phenotype that arises as a novel pathological mechanism underlying the severe neurological defects present in patients. In view of this, astrocytes appear as new therapeutic targets for AHDS. Declarations Ethics approval and consent to participate The processing and use of the human tissue samples were approved by the ethics committee of Consejo Superior de Investigaciones Científicas (CSIC; numbers of permission: 045/2019 for SAF2017-86342-R and 124/2022 for PID2020-113139RB-I00). Human samples and data were obtained with informed consent from the families, in agreement with the Declaration of Helsinki. Terms and conditions to analyze the human brain MRI images were approved by an Authorized Official of both Institutions: The Medical University of South Carolina and Instituto de Investigaciones Biomédicas Sols-Morreale by means of a Data Transfer and Use Agreement. In all cases, personal data were treated anonymously. All experimental procedures involving animals were performed following the European Union Council guidelines (directive 2010/63/UE) and Spanish regulations (R.D. 53/2013) and were approved by the ethics committee Comité de Ética y Experimentación Humana y Animal (CEEHA) and by the Comunidad Autónoma de Madrid Review Board for the use of animals for scientific purposes (approval numbers 162/17, 252.7/20 and 014.1/21). Availability of data and materials The datasets supporting the conclusions of this article are included within the article and its additional files. Competing interests The authors declare that they have no competing interests. Acknowledgements We are extremely grateful to the patients’ parents who gave their consent to use the brain tissues and the MR images for this investigation. We are also indebted to the IdiPAZ Biobank (PT20/00004) and the Sant Joan de Déu Hospital Biobank integrated into the Spanish National Biobanks and the Sydney Children’s and Prince of Wales Hospitals, Randwick, Australia as well as the Wolfson Medical Center, Holon, Israel, and the Sackler School of Medicine, Tel Aviv, Israel, for the generous gifts of clinical samples used in this work. We thank Daniela López-Espíndola for her contribution to the fetal brain initial studies and Estrella Rausell, Laura Barrios and Belén Garzón for their helpful advice. We would like to thank María Camino de Lucas, Cristina Crespo Fernández, and Coral Pedrero García for animal care. Funding This study was supported by 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 (Grant No. 2020AEP044 to AG-F), The Sherman Foundation (Grant No. OTR02211 to SB-L and AG-F), Asociación Corriendo con el Corazón por Hugo (Grant No. OTR06190 to AG-F), a contract from Ministerio de Ciencia, Innovación y Universidades (Programa de Formación de Profesorado (FPU, FPU19/02006) to MG-Y, a contract from Universidad Autónoma de Madrid (Contrato predoctoral para Formación de personal Investigador en Formación, FPI-UAM) to AG-A, a contract from MCIN/AEI/10.13039/501100011033 and “ESF Investing in your future” (Grant No. PRE2018-086185) to VV-H, and a grant from the MCIN/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTR (Grant No. IJC2020-043543-I) to SB-L. Authors and affiliations Laboratory of Thyroid hormones and CNS. Department of Neurological Diseases and Aging. Instituto de Investigaciones Biomédicas Sols-Morreale, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), Madrid, Spain. Marina Guillén-Yunta, Ángel García-Aldea, Víctor Valcárcel-Hernández, Ainara Sanz-Bógalo, Carmen Grijota-Martínez, Soledad Bárez-López, Ana Montero-Pedrazuela & Ana Guadaño-Ferraz. Magnetic Imaging Resonance Core Facility, Institut d'Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), Barcelona, Spain. Emma Muñoz-Moreno. Department of Radiology and Radiological Science, Medical University of South Carolina, Charleston, SC, USA. Maria Gisele Matheus. Department of Cell Biology, Faculty of Biology, Universidad Complutense de Madrid, Madrid, Spain. Carmen Grijota-Martínez Authors’ contributions MG-Y, AG-A, CG-M, SB-L, AM-P and AG-F conceived and planed the experimental procedures. MG-Y, ÁG-A, VV-H, AS-B, CG-M, SB-L and AM-P carried out the experiments and took care of the colony at our animal facility. MG-Y, SB-L and AM-P quantified and analyzed data obtained from the experimental procedures. MG-M provided the MRI images from control and MCT8-deficient subjects and EM-M segmented and quantified the ADC values. AG-F supervised and administered the research project. SB-L wrote the original manuscript with the support from MG-Y, AM-P and AG-F. All authors read and approved the final manuscript. References Allan W, Herndon C, Dudley FC. Some examples of the inheritance of mental deficiency: apparently sex-linked idiocy and microcephaly. Am J Ment Defic. 1944;48(48):325-34. Dumitrescu AM, Liao X-H, Best TB, Brockmann K, Refetoff S. A Novel Syndrome Combining Thyroid and Neurological Abnormalities Is Associated with Mutations in a Monocarboxylate Transporter Gene. The American Journal of Human Genetics. 2004;74(1):168-75. Friesema ECH, Grueters A, Biebermann H, Krude H, von Moers A, Reeser M, et al. Association between mutations in a thyroid hormone transporter and severe X-linked psychomotor retardation. The Lancet. 2004;364(9443):1435-7. Friesema EC, Ganguly S, Abdalla A, Manning Fox JE, Halestrap AP, Visser TJ. Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter. J Biol Chem. 2003;278(41):40128-35. Masnada S, Sarret C, Antonello CE, Fadilah A, Krude H, Mura E, et al. Movement disorders in MCT8 deficiency/Allan-Herndon-Dudley Syndrome. Mol Genet Metab. 2022;135(1):109-13. Remerand G, Boespflug-Tanguy O, Tonduti D, Touraine R, Rodriguez D, Curie A, et al. Expanding the phenotypic spectrum of Allan–Herndon–Dudley syndrome in patients with SLC16A2 mutations. Developmental Medicine & Child Neurology. 2019;61(12):1439-47. Matheus MG, Lehman RK, Bonilha L, Holden KR. Redefining the Pediatric Phenotype of X-Linked Monocarboxylate Transporter 8 (MCT8) Deficiency: Implications for Diagnosis and Therapies. J Child Neurol. 2015;30(12):1664-8. Ceballos A, Belinchon MM, Sanchez-Mendoza E, Grijota-Martinez C, Dumitrescu AM, Refetoff S, et al. Importance of monocarboxylate transporter 8 for the blood-brain barrier-dependent availability of 3,5,3'-triiodo-L-thyronine. Endocrinology. 2009;150(5):2491-6. López-Espíndola D, García-Aldea Á, Gómez de la Riva I, Rodríguez-García AM, Salvatore D, Visser TJ, et al. Thyroid hormone availability in the human fetal brain: novel entry pathways and role of radial glia. Brain Struct Funct. 2019;224(6):2103-19. Roberts LM, Woodford K, Zhou M, Black DS, Haggerty JE, Tate EH, et al. Expression of the thyroid hormone transporters monocarboxylate transporter-8 (SLC16A2) and organic ion transporter-14 (SLCO1C1) at the blood-brain barrier. Endocrinology. 2008;149(12):6251-61. López-Espíndola D, Morales-Bastos C, Grijota-Martínez C, Liao XH, Lev D, Sugo E, et al. Mutations of the thyroid hormone transporter MCT8 cause prenatal brain damage and persistent hypomyelination. J Clin Endocrinol Metab. 2014;99(12):E2799-804. Vancamp P, Demeneix BA, Remaud S. Monocarboxylate Transporter 8 Deficiency: Delayed or Permanent Hypomyelination? Front Endocrinol (Lausanne). 2020;11:283. Valcárcel-Hernández V, López-Espíndola D, Guillén-Yunta M, García-Aldea Á, López de Toledo Soler I, Bárez-López S, et al. Deficient thyroid hormone transport to the brain leads to impairments in axonal caliber and oligodendroglial development. Neurobiol Dis. 2022;162:105567. Guillén-Yunta M, Valcárcel-Hernández V, García-Aldea Á, Soria G, García-Verdugo JM, Montero-Pedrazuela A, et al. Neurovascular unit disruption and blood-brain barrier leakage in MCT8 deficiency. Fluids Barriers CNS. 2023;20(1):79. Manzano J, Bernal J, Morte B. Influence of thyroid hormones on maturation of rat cerebellar astrocytes. Int J Dev Neurosci. 2007;25(3):171-9. Dezonne RS, Stipursky J, Gomes FC. Effect of thyroid hormone depletion on cultured murine cerebral cortex astrocytes. Neurosci Lett. 2009;467(2):58-62. Morte B, Gil-Ibáñez P, Bernal J. Regulation of Gene Expression by Thyroid Hormone in Primary Astrocytes: Factors Influencing the Genomic Response. Endocrinology. 2018;159(5):2083-92. Bernal J, Morte B, Diez D. Thyroid hormone regulators in human cerebral cortex development. J Endocrinol. 2022;255(3):R27-r36. Wang T, Wang Y, Montero-Pedrazuela A, Prensa L, Guadaño-Ferraz A, Rausell E. Thyroid Hormone Transporters MCT8 and OATP1C1 Are Expressed in Projection Neurons and Interneurons of Basal Ganglia and Motor Thalamus in the Adult Human and Macaque Brains. Int J Mol Sci. 2023;24(11). Bernal J, Guadaño-Ferraz A, Morte B. Thyroid hormone transporters--functions and clinical implications. Nat Rev Endocrinol. 2015;11(7):406-17. Guadaño-Ferraz A, Obregón MJ, St Germain DL, Bernal J. The type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain. Proc Natl Acad Sci U S A. 1997;94(19):10391-6. Niedowicz DM, Wang WX, Price DA, Xie K, Patel E, Nelson PT. Impact of thyroid hormone perturbations in adult mice: brain weight and blood vessel changes, gene expression variation, and neurobehavioral outcomes. Neurobiol Aging. 2023;128:74-84. Mayerl S, Heuer H. Thyroid hormone transporter Mct8/Oatp1c1 deficiency compromises proper oligodendrocyte maturation in the mouse CNS. Neurobiol Dis. 2023;184:106195. Chamas L, Seugnet I, Poirier R, Clerget-Froidevaux MS, Enderlin V. A Fine Regulation of the Hippocampal Thyroid Signalling Protects Hypothyroid Mice against Glial Cell Activation. Int J Mol Sci. 2022;23(19). Matusova Z, Hol EM, Pekny M, Kubista M, Valihrach L. Reactive astrogliosis in the era of single-cell transcriptomics. Front Cell Neurosci. 2023;17:1173200. Muñoz-Ballester C, Robel S. Astrocyte-mediated mechanisms contribute to traumatic brain injury pathology. WIREs Mech Dis. 2023:e1622. Sofroniew MV. Astrogliosis. Cold Spring Harb Perspect Biol. 2014;7(2):a020420. Voorn JPvd, Pouwels PJW, Powers JM, Kamphorst W, Martin J-J, Troost D, et al. Correlating Quantitative MR Imaging with Histopathology in X-Linked Adrenoleukodystrophy. American Journal of Neuroradiology. 2011;32(3):481-9. Lodygensky GA, West T, Stump M, Holtzman DM, Inder TE, Neil JJ. In vivo MRI analysis of an inflammatory injury in the developing brain. Brain, Behavior, and Immunity. 2010;24(5):759-67. Bárez-López S, Grijota-Martínez C, Ausó E, Fernández-de Frutos M, Montero-Pedrazuela A, Guadaño-Ferraz A. Adult Mice Lacking Mct8 and Dio2 Proteins Present Alterations in Peripheral Thyroid Hormone Levels and Severe Brain and Motor Skill Impairments. Thyroid. 2019;29(11):1669-82. Valcárcel-Hernández V, Guillén-Yunta M, Bueno-Arribas M, Montero-Pedrazuela A, Grijota-Martínez C, Markossian S, et al. A CRISPR/Cas9-engineered avatar mouse model of monocarboxylate transporter 8 deficiency displays distinct neurological alterations. Neurobiol Dis. 2022;174:105896. Avants BB, Epstein CL, Grossman M, Gee JC. Symmetric diffeomorphic image registration with cross-correlation: evaluating automated labeling of elderly and neurodegenerative brain. Med Image Anal. 2008;12(1):26-41. Fischl B, Salat DH, Busa E, Albert M, Dieterich M, Haselgrove C, et al. Whole brain segmentation: automated labeling of neuroanatomical structures in the human brain. Neuron. 2002;33(3):341-55. Dumitrescu AM, Liao XH, Weiss RE, Millen K, Refetoff S. Tissue-specific thyroid hormone deprivation and excess in monocarboxylate transporter (mct) 8-deficient mice. Endocrinology. 2006;147(9):4036-43. Schneider MJ, Fiering SN, Pallud SE, Parlow AF, St Germain DL, Galton VA. Targeted disruption of the type 2 selenodeiodinase gene (DIO2) results in a phenotype of pituitary resistance to T4. Mol Endocrinol. 2001;15(12):2137-48. Dutta S, Sengupta P. Men and mice: Relating their ages. Life Sci. 2016;152:244-8. Morel L, Men Y, Chiang MSR, Tian Y, Jin S, Yelick J, et al. Intracortical astrocyte subpopulations defined by astrocyte reporter Mice in the adult brain. Glia. 2019;67(1):171-81. Akdemir ES, Huang AY, Deneen B. Astrocytogenesis: where, when, and how. F1000Res. 2020;9. Lattke M, Guillemot F. Understanding astrocyte differentiation: Clinical relevance, technical challenges, and new opportunities in the omics era. WIREs Mech Dis. 2022;14(5):e1557. Kantarci K, Petersen RC, Boeve BF, Knopman DS, Weigand SD, O'Brien PC, et al. DWI predicts future progression to Alzheimer disease in amnestic mild cognitive impairment. Neurology. 2005;64(5):902-4. Iwayama H, Liao XH, Braun L, Bárez-López S, Kaspar B, Weiss RE, et al. Adeno Associated Virus 9-Based Gene Therapy Delivers a Functional Monocarboxylate Transporter 8, Improving Thyroid Hormone Availability to the Brain of Mct8-Deficient Mice. Thyroid. 2016;26(9):1311-9. Vatine GD, Al-Ahmad A, Barriga BK, Svendsen S, Salim A, Garcia L, et al. Modeling Psychomotor Retardation using iPSCs from MCT8-Deficient Patients Indicates a Prominent Role for the Blood-Brain Barrier. Cell Stem Cell. 2017;20(6):831-43.e5. Yamaguchi M, Seki T, Imayoshi I, Tamamaki N, Hayashi Y, Tatebayashi Y, et al. Neural stem cells and neuro/gliogenesis in the central nervous system: understanding the structural and functional plasticity of the developing, mature, and diseased brain. J Physiol Sci. 2016;66(3):197-206. Gil-Ibañez P, García-García F, Dopazo J, Bernal J, Morte B. Global Transcriptome Analysis of Primary Cerebrocortical Cells: Identification of Genes Regulated by Triiodothyronine in Specific Cell Types. Cereb Cortex. 2017;27(1):706-17. Perez-Catalan NA, Doe CQ, Ackerman SD. The role of astrocyte-mediated plasticity in neural circuit development and function. Neural Dev. 2021;16(1):1. Rakic P. Mode of cell migration to the superficial layers of fetal monkey neocortex. J Comp Neurol. 1972;145(1):61-83. Mohan V, Sinha RA, Pathak A, Rastogi L, Kumar P, Pal A, et al. Maternal thyroid hormone deficiency affects the fetal neocorticogenesis by reducing the proliferating pool, rate of neurogenesis and indirect neurogenesis. Exp Neurol. 2012;237(2):477-88. Supplementary Files GuillenYuntaetal.Supplementarydata.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":268429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBrain Apparent Diffusion Coefficient (ADC) analysis in MCT8-deficient patients. a-b. \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003eRegions of Interest (ROIs) used for quantitative analysis of ADC values. Axial (a) and coronal (b) sections of the brain from a control subject are displayed, featuring representative delineated ROIs for the cerebral cortex (in dark blue) and striatum (in cyan). While unilateral ROIs are presented for clarity, bilateral ROIs were employed in the subsequent analytical procedures.\u003cstrong\u003e c-d. \u003c/strong\u003eDot plots show the distribution of individual ADC values by age in the cerebral cortex and striatum, respectively.\u003cstrong\u003e e-f. \u003c/strong\u003eAnalysis of the ADC value in the cerebral cortex (e) and striatum (f) in the brain of MCT8-deficient patients (red dots, n = 11) and age-matched controls (blue dots, n = 10-11). The mean ADC value and SD are shown. ****p \u0026lt; 0.0001 was determined by General Linear Model with one fixed factor (Control, MCT8-deficient) and age as a covariate.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3825537/v1/2f65bfa1e88510dd8cd4fd6d.png"},{"id":50441111,"identity":"222409e7-8b0e-444b-85b0-f24f934bdc6c","added_by":"auto","created_at":"2024-01-31 15:16:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13401287,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGFAP immunolabeling in the human motor cortex. \u003c/strong\u003eExpression of GFAP in the motor cortex of a 12-year-old control subject and an 11-year-old MCT8-deficient subject. \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003eb.\u003c/strong\u003e Panoramic view of the gyrus of the motor cortex, including all layers and the subcortical WM. Note the absence of staining in layers II-VI of the control sample and the widespread GFAP expression in the MCT8-deficient sample. \u003cstrong\u003ec-l.\u003c/strong\u003eHigh-magnification images of different cortical layers and the subcortical WM. I-VI: layers of the motor cortex. WM: white matter. Scale bar: 1,000 μm in a-b; 50 μm in c-l.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3825537/v1/fd67472086646708c203542f.png"},{"id":50441114,"identity":"a9e750f6-88cc-431b-bebc-09b7e9b8329c","added_by":"auto","created_at":"2024-01-31 15:16:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4853856,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGFAP immunolabeling in the human striatum.\u003c/strong\u003e \u003cstrong\u003ea,c.\u003c/strong\u003eLow magnification image (a) and high magnification inset (c) of the expression of GFAP in the striatum of a 10-year-old control subject \u003cstrong\u003eb,d.\u003c/strong\u003e Low magnification image (b) and high magnification inset (d) of the expression of GFAP in the striatum of an 11-year-old MCT8-deficient subject. Scale bar: 50 μm in a-b; 20 μm in c-d.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3825537/v1/feb2dbfbb715d2ab4422edba.png"},{"id":50444131,"identity":"8f9a90e7-f4ef-4a52-95dc-089aa9a017bb","added_by":"auto","created_at":"2024-01-31 15:32:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13289379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGFAP and ALDH1L1 immunolabeling in the motor cortex of WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMct8/Dio2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eKO mice. \u003c/strong\u003eAnalysis performed using 3-month-old WT (n = 4) and \u003cem\u003eMct8/Dio2\u003c/em\u003eKO (n = 4) mice. \u003cstrong\u003ea-f. \u003c/strong\u003eGFAP expression. \u003cstrong\u003eg-l. \u003c/strong\u003eALDH1L1\u003cstrong\u003e \u003c/strong\u003eexpression. a,b, and g,h show the general distribution pattern of GFAP+ and ALDH1L1+\u003cstrong\u003e \u003c/strong\u003eastrocytes, respectively. Higher magnification images show details in layer I (c-d for GFAP, i-j for ALDH1L1) and layers IV-V (e-f for GFAP and k-l for ALDH1L1). Observe the different GFAP expression pattern in the inner layers of the cortex between both genotypes (e, f). Scale bar: 125 μm in a-b and g-h; 20 μm in c-f and i-l. \u003cstrong\u003em-n.\u003c/strong\u003e Quantitative analysis of the density of GFAP+ (m) and ALDH1L1+ (n) astrocytes in the M2 of WT (blue) and \u003cem\u003eMct8/Dio2\u003c/em\u003eKO (red) mice. ****p \u0026lt; 0.0001 was determined by a two-tailed unpaired Student’s t-test. M2: secondary motor cortex. FC: fold change.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3825537/v1/e6ba6ba2854815c4c8650907.png"},{"id":50443029,"identity":"8290f6a8-086d-4983-983b-784ca4fc5bb3","added_by":"auto","created_at":"2024-01-31 15:24:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2338658,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGFAP and ALDH1L1 immunolabeling in the striatum of WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMct8/Dio2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eKO mice. \u003c/strong\u003eAnalysis performed using 3-month-old WT (n = 4) and \u003cem\u003eMct8/Dio2\u003c/em\u003eKO (n = 4) mice. \u003cstrong\u003ea-d. \u003c/strong\u003eGFAP expression. \u003cstrong\u003ee-h. \u003c/strong\u003eALDH1L1\u003cstrong\u003e \u003c/strong\u003eexpression. a,b, and e,f show the general distribution pattern of GFAP+ and ALDH1L1+\u003cstrong\u003e \u003c/strong\u003eastrocytes, respectively, in the striatum. No differences were observed with any of the markers between genotypes. Higher magnification images show details (c-d for GFAP, g-h for ALDH1L1). Scale bar: 200 μm in a-b and e-f; 20 μm in c-d and g-h.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3825537/v1/ba36e23115ebe8a779c0c1b0.png"},{"id":50441113,"identity":"5090ae39-18f9-44a5-8b33-8406047d0f05","added_by":"auto","created_at":"2024-01-31 15:16:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5682224,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGFAP immunolabeling in the human developing occipital cortex.\u003c/strong\u003e Expression of GFAP in the occipital cortex of a 30th gestational week control subject and an MCT8-deficient subject. \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003eb.\u003c/strong\u003e Panoramic view of the occipital cortex, including all layers and the subcortical WM. \u003cstrong\u003ec-l.\u003c/strong\u003e High-magnification images of the different cortical layers and the subcortical WM. Note the reduction in GFAP+ radial glial processes in layer I and WM in the MCT8-deficient sample as compared to the control sample. I-VI: layers of the motor cortex. WM: white matter. Scale bar: 310 μm in a-b; 31 μm in c-l.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3825537/v1/39a38b2bfd0ced0b6425b5b6.png"},{"id":50441112,"identity":"e4b25853-fffb-460c-8f18-2b730d402e92","added_by":"auto","created_at":"2024-01-31 15:16:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5224637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNissl staining and GFAP immunolabeling in the developing visual cortex of WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMct8/Dio2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eKO mice.\u003c/strong\u003e Analysis performed using P7 and P15 WT and \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice (n = 4 for each genotype and age). \u003cstrong\u003ea-d. \u003c/strong\u003eImage composition of\u003cstrong\u003e \u003c/strong\u003eNissl staining of the visual cortex of P7 (a-b) and P15 (c-d) mice showing the cell distribution and cortical lamination. Note the different cell density in supra- and infragranular layers at P7 between genotypes. \u003cstrong\u003ee-l.\u003c/strong\u003e GFAP expression in the visual cortex of P7 (e-h) and P15 (i-l) mice. Note that \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice show altered GFAP+ radial glial cells at P7 as compared to WT and the pronounced increase of GFAP+ astrocytes at P15 suggesting astrogliosis. P: postnatal day. I-VI: layers of the motor cortex. WM: white matter. Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3825537/v1/ba921f7e4be7f2abd53e8274.png"},{"id":50824641,"identity":"2d51f0d2-072d-491e-adff-6c4c0ed7a238","added_by":"auto","created_at":"2024-02-07 23:08:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12184641,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3825537/v1/b4bc6bf4-664e-49b5-a585-e0aa4f2ba7e5.pdf"},{"id":50441117,"identity":"5f10c8f4-aa92-47f1-8019-64c3065d3af6","added_by":"auto","created_at":"2024-01-31 15:16:45","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5952809,"visible":true,"origin":"","legend":"","description":"","filename":"GuillenYuntaetal.Supplementarydata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3825537/v1/683326bc00aca8bcf6dbdb95.pdf"}],"financialInterests":"","formattedTitle":"Defective thyroid hormone transport to the brain leads to astroglial alterations","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eAllan-Herndon-Dudley Syndrome (AHDS) or MCT8 deficiency is a rare X-linked disorder caused by inactivating mutations in the gene that codifies for the monocarboxylate transporter 8 (MCT8) (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). MCT8 is a transmembrane transporter highly specific for thyroid hormones (THs), both thyroxine (T4) and the transcriptionally active form 3,5,3\u0026rsquo;-triiodothyronine (T3) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). This syndrome is characterized by peripheral hyperthyroidism concomitant with brain hypothyroidism. Patients also present severe neurological damage, psychomotor retardation, and global developmental delay. Moreover, the majority of affected boys have profound intellectual disability (IQ\u0026thinsp;\u0026lt;\u0026thinsp;30), speech difficulties, as well as central hypotonia, spastic paraplegia, and dystonic movements (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The neurological impairments have been linked to impaired transport of THs across the brain barriers (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), where MCT8 is highly expressed in humans (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), leading to low T3 and T4 content in the brain (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). However, despite these insights, the neuropathophysiological mechanisms underlying MCT8 deficiency are not well understood and there is no effective treatment to palliate the severe neurological impairments in MCT8-deficient patients.\u003c/p\u003e \u003cp\u003eIn order to develop therapeutic approaches to improve the neurological alterations in MCT8-deficient patients, it is essential to have a complete understanding of the nature of these alterations. The defects identified to date in the brain of MCT8-deficient patients are limited and point to a wide range of alterations including deficient myelination (reviewed in (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)), decreased myelinated axonal diameter (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), altered cortical and cerebellar structure, defects at the synapse with deficient expression of synaptophysin, defects at GABAergic interneurons with abnormal parvalbumin and calbindin-D28k expression (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), as well as neurovascular unit disruption (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The complex neurological phenotype in MCT8-deficient patients seems to be arising from several brain impairments. The identification of additional pathological mechanisms mediating the brain alterations of patients will provide valuable information to design appropriate treatments.\u003c/p\u003e \u003cp\u003eTHs have been shown to regulate the maturation and function of astrocytes both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Astroglial cells express TH receptors (TRs) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), indicating that they are direct targets of THs action (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Indeed, astrocytes express the MCT8 transporter in human (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), monkey (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), and mouse (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Astrocytes also express the enzyme deiodinase 2 (DIO2) which locally generates the main cerebral pool of T3 from T4 (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). There is recent evidence suggesting that brain hypothyroidism leads to astrogliosis (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Astrogliosis is a reaction of astrocytes to disturbed homeostasis in the brain, with changes in the number of astrocytes, their morphology, and function. While changes in astrocytes might initiate repair processes in the brain, they can also be detrimental by leading to secondary damage such as neuronal death or abnormal neuronal activity (\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn view of this, we hypothesized that MCT8 deficiency affects the astroglial population which may be one of the underlying causes for some of the neurological defects in MCT8-deficient patients. To test this hypothesis, we analyzed apparent diffusion coefficient (ADC) imaging values from MCT8-deficient patients and we identified histopathological abnormalities associated with altered cytoarchitecture, including gliosis (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Alterations in astroglia were confirmed by immunolabeling brain samples of an 11-year-old and a 30th gestational week MCT8-deficient subjects with antibodies against astroglial markers. These findings were validated and further explored in a mouse model of AHDS. Our findings confirm changes in the astroglial population in MCT8 deficiency that arise early in brain development and persist at adult stages, revealing an abnormal distribution, density, and morphology of cortical astrocytes, compatible with an astrogliosis-like phenotype at adult stages.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eStudy design\u003c/h2\u003e\n \u003cp\u003eThis study examined brain cytoarchitecture alterations, in particular in the astroglia population, as a pathophysiological mechanism mediating the severe brain impairments in MCT8 deficiency. First, we calculated the ADC imaging values obtained from magnetic resonance imaging in the cerebral cortex and the striatum of 11 MCT8-deficient patients and 11 control subjects in a single-blinded fashion. Next, we further studied potential brain alterations by histochemistry and immunohistochemistry in autopsy brain samples from an 11-year-old and a 30th gestational week MCT8-deficient subjects in comparison to brain samples from control subjects at similar ages. In addition, we studied brain samples from a previously validated mouse model of the AHDS (\u003cem\u003eMct8/Dio2\u003c/em\u003eKO, (\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e)). Data from control and \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice were acquired in a single-blinded manner with a number assigned to each animal unrelated to their genotype. Sample size for immunohistochemistry (n\u0026thinsp;=\u0026thinsp;4) was selected according to previous experience (\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eMagnetic resonance imaging acquisition and processing\u003c/h2\u003e\n \u003cp\u003eBrain MR Imaging (MRI) acquisition was performed in a cohort of 22 boys between 3 and 13 years old including 11 MCT8-deficient patients and 11 age-matched controls (see Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) using a 3.0T scanner (Magnetom Skyra-Siemens Heathinners, Erlangen, Germany) at the Medical University of South Carolina. The examination included T1-weighted images acquired with an Inversion Recovery sequence (TE\u0026thinsp;=\u0026thinsp;2.5 ms, TR\u0026thinsp;=\u0026thinsp;1900 ms, TI\u0026thinsp;=\u0026thinsp;900 ms, matrix 256x256, voxel-size\u0026thinsp;=\u0026thinsp;0.86x0.86x0.86 mm\u0026sup3;) and diffusion-weighted imaging (DWI) with two b-values: b\u0026thinsp;=\u0026thinsp;0 and b\u0026thinsp;=\u0026thinsp;1000 s/mm\u0026sup2; to estimate ADC maps (TR\u0026thinsp;=\u0026thinsp;6400ms, TE\u0026thinsp;=\u0026thinsp;98ms, voxel-size\u0026thinsp;=\u0026thinsp;1.2x1.2x5mm\u0026sup3;).\u003c/p\u003e\n \u003cp\u003eDWI images were processed to obtain the ADC maps. First, elastic registration to T1-weighted images was performed to remove EPI distortion using ANTs (\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e), and ADC was estimated from the images acquired with b\u0026thinsp;=\u0026thinsp;0 and b\u0026thinsp;=\u0026thinsp;1000 s/mm\u0026sup2;. T1-weigthed images were processed with Freesurfer 7.1.1 software (\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e) to obtain brain parcellation into anatomical regions. This parcellation was then translated to the ADC map, and average ADC value in the regions of interest was obtained. Based in the psychomotor impairments and profound intellectual disability of the patients, the regions of interest were the cerebral cortex (left and right) and striatum (including caudate and putamen regions identified by Freesurfer).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eFeatures of the subjects enrolled in Magnetic Resonance Imaging (MRI) studies.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSubject\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDiagnosis\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAge (y)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eHuman tissue samples\u003c/h2\u003e\n \u003cp\u003eAs in L\u0026oacute;pez-Esp\u0026iacute;ndola et al. (\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e), we used postmortem brain paraffin samples from an 11-year-old subject with a mutation (Q96X) in the \u003cem\u003eSLC16A2\u003c/em\u003e gene, with severe psychomotor impairment, high serum T3, low rT3 and T4, and mildly elevated TSH. Previously reported MRI showed a mildly delayed myelination at 9 months, along with a slight dilatation of the lateral ventricles\u0026rsquo; frontal horns at 6.5 years. The cause of death was a respiratory failure secondary to aspiration pneumonia. The paraffin blocks were provided by the Sydney Children\u0026rsquo;s and Prince of Wales Hospitals, Randwick, Australia. Control samples came from a 10-year-old girl and a 12-year-old boy whose causes of death were acute pulmonary edema during a surgical procedure and diffuse lymphocytic myocarditis, respectively. Paraffin blocks were kindly provided by the IdiPAZ Biobank (PT20/00004), and the Biobanc per a la Investigaci\u0026oacute; de l\u0026rsquo;Hospital Infantil Sant Joan de D\u0026eacute;u, Barcelona (integrated into the Spanish National Biobanks Network) and they were processed following standard operating procedures with the appropriate approval of the Ethics and Scientific Committees.\u003c/p\u003e\n \u003cp\u003eAs in L\u0026oacute;pez-Esp\u0026iacute;ndola et al. (\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e), we also used a 30th gestational week male fetus with a mutation (L494P) in the \u003cem\u003eSLC16A2\u003c/em\u003e gene identified by amniocentesis. Pregnancy was terminated upon parental request and approval by an officially designated committee (Wolfson Medical Center, Holon, Israel, and the Sackler School of Medicine, Tel Aviv, Israel) and paraffin blocks were provided by the Wolfson Medical Center. As a control, a 30th gestational week male fetus aborted due to placental abruption was used. Paraffin blocks were provided by the IdiPAZ Biobank (PT20/00004).\u003c/p\u003e\n \u003cp\u003eAll paraffin blocks were sliced in a microtome (Microm, HM 310) at 7 \u0026micro;m of thickness for immunohistochemistry assays.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eExperimental animals\u003c/h2\u003e\n \u003cp\u003eAnimals were housed in temperature- and light-controlled conditions at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C on a 12:12 light-dark cycle (lights on at 7 AM), with access to food and water \u003cem\u003ead libitum\u003c/em\u003e. Since AHDS is an X-linked disease affecting almost exclusively males, only wild-type (WT) and \u003cem\u003eMct8\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/y\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/Dio2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eMct8/Dio2\u003c/em\u003eKO) male mice were used in all studies. Animals were euthanized at postnatal day 7 (P7), P15, 3, and 6 months of age for immunohistochemical analysis. \u003cem\u003eMct8\u003c/em\u003eKO mice were originally produced by Dumitrescu et al. 2006 (\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e) and \u003cem\u003eDio2\u003c/em\u003eKO by Schneider et al. 2001 (\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e). \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice were bred at the animal facility of the Instituto de Investigaciones Biom\u0026eacute;dicas Sols-Morreale by crossing \u003cem\u003eMct8\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/y\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/Dio2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e males and \u003cem\u003eMct8\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/Dio2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e females. Animals were housed in groups of up to 6 mice per cage with environmental enrichment consisting of nesting material and a cardboard tube. All genotypes were confirmed by PCR of ear DNA as described (\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eFor histological procedures, mice were anesthetized with ketamine (75 \u0026micro;g/g of body weight) and medetomidine hydrochloride (1 \u0026micro;g/g of body weight) and transcardially perfused with 4% paraformaldehyde in 0.1M phosphate buffer (PB). Brains were removed, post-fixed overnight in 4% paraformaldehyde in 0.1M PB, cryoprotected in 30% sucrose, and cut into 25 \u0026micro;m coronal sections on a cryostat. All sample collections were performed between 09.00 and 12.00 a.m. with time-matched experimental controls run in parallel.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunohistochemistry and histology\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003eHuman brain samples\u003c/strong\u003e: Immunohistochemical procedures for human brain samples were performed as previously described (\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e). Tissue sections were deparaffinized and re-hydrated. To facilitate the antigen-antibody reaction, sections were incubated for 20 min at 95 \u0026deg;C with the Envision FLEX Low pH solution for antigen retrieval (DAKO, K8005). Endogenous peroxidase was blocked by using 3% hydrogen peroxide in distilled water for 15 min. Samples were blocked with phosphate-buffered saline (PBS) containing 4% bovine serum albumin (BSA, Sigma A4503), 0.1% Triton X-100, 0.1 M Lysine, and 5% normal goat serum (Vector Laboratories, S-1000) for 1 h. Samples were incubated overnight at 4 \u0026deg;C with the anti-Glial Fibrillary Acidic Protein (GFAP) antibody (1:200, DAKO Z0334) in PBS containing 4% BSA, 0.1% Triton X-100, and 1% normal goat serum. Tissues were washed in PBS and incubated for 1 h at RT with the corresponding biotinylated secondary antibody (Vector Laboratories) in PBS containing 4% BSA, 0.1% Triton, and 1% serum. In order to amplify the immune signal, tissue samples were incubated for 1 h in the dark with Avidin-Biotin-Complex (ABC Elite Kit; Vector Laboratories, #32050), according to the manufacturer\u0026rsquo;s instructions. Finally, tissue sections were incubated with diaminobenzidine (0.5 mg/mL, Sigma, D5637) in 0.01% hydrogen peroxide. Preparations from the 30th gestational week male fetuses were counterstained with Harris hematoxylin for a few seconds (Sigma, HHS32). The sections were dehydrated, cleared in xylene, and cover-slipped with DePeX (Serva, 18243). Negative controls omitting the primary antibody run in parallel displayed no immunopositive signal.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMouse brain samples\u003c/strong\u003e: The immunodetection was performed in free-floating sections as previously described (\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e). For Aldehyde Dehydrogenase 1 Family Member L1 (ALDH1L1) immunolabelling, sections were incubated in Envision FLEX Low pH solution for antigen retrieval buffer (DAKO, K8005) for 12 min at 95\u0026deg;C. This step was omitted for GFAP immunostaining. After several washes in PBS, the endogenous peroxidase activity was blocked using 10% methanol and 3% hydrogen peroxide in PBS for 15 min in the dark. Immunolabeling using anti-GFAP (1:3000, DAKO Z0334) and ALDH1L1 (1:1000, Proteintech 17390-1-AP) was performed as described for human samples. Genotypes and time-matched experimental controls were run in parallel to avoid methodological differences. Negative controls omitting the primary antibody, also run in parallel, displayed no immunopositive signal.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eImage analysis and quantification\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eImmunohistochemically-labeled brain sections were examined under brightfield illumination using a Nikon Eclipse 80i (Nikon Corp., Tokyo, Japan) microscope and microphotographs were taken with a Nikon DSFi1 digital camera. Cortical lamination was determined by analyzing parallel tissue sections stained with hematoxylin-eosin in human and Nissl staining in mouse. Quantification of GFAP immunopositive (GFAP+) cell density in the mouse motor cortex was evaluated by counting all the GFAP\u0026thinsp;+\u0026thinsp;cells in the secondary motor cortex (M2) in four sections between bregma 1.18 to -0.58 in four different animals per genotype. M2 cortex (layers I to VI) was delineated using a 20x (numerical aperture 0.5) and GFAP\u0026thinsp;+\u0026thinsp;cells were counted using a 40x objective (numerical aperture 0.75).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistics\u003c/h2\u003e\n \u003cp\u003eData were expressed in box and whisker plots. Statistical analyses were performed using GraphPad Prism Software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.graphpad.com\u003c/span\u003e\u003c/span\u003e). Normality of the data was assessed by the Shapiro\u0026ndash;Wilk test. Means between two groups were compared with a 2-tailed unpaired Student\u0026rsquo;s t-test for parametric data and with a 2-tailed Mann Whitney test for non-parametric data. Significant differences were represented as * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. In human MRI studies, the effect of age and condition on ADC values was estimated using a General Linear Model with one fixed factor (Control, MCT8-deficient) and age as a covariate. Pearson\u0026rsquo;s correlation coefficient was used to explore the statistical relationship between the cerebral cortex and the striatum. Data are presented in box-plots representing the 25th (bottom), 50th (middle-line) and 75th (top) quartiles with whiskers extending from minimum to maximum values, otherwise specified.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBrain water diffusivity is altered in MCT8-deficient patients\u003c/h2\u003e \u003cp\u003eThe brains from 11 MCT8-deficient patients and 11 age-matched controls ranging from 3 to 13 years old were non-invasively studied using brain DWI. DWI allows the assessment of the water ADC, a measurement of tissue water diffusivity which is altered in different pathological conditions. Changes in the ADC value have been associated with alterations in brain cytoarchitecture, including gliosis (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe analysis of ADC was focused on the cerebral cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), a brain region involved in high-order cognitive processing, and the striatum, a brain structure associated with movement disorders (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cb\u003ed\u003c/b\u003e depict each individual ADC value \u003cem\u003eversus\u003c/em\u003e the age of the corresponding patient in the cerebral cortex and striatum, respectively. We observed that the ADC value did not change with age in either MCT8-deficient or control subjects in the cerebral cortex and the striatum. Most importantly, we observed a significant increase in ADC values in MCT8-deficient patients compared to controls in both the cerebral cortex (F(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;29.339, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) and the striatum F(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;33.665, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMCT8 deficiency leads to persistent astroglial alterations in the cerebral cortex\u003c/h2\u003e \u003cp\u003eTo assess whether the increased ADC values in MCT8-deficient patients were associated to astroglial alterations, we evaluated the density and morphology of astroglial populations in brain samples of an 11-year-old MCT8-deficient patient. This was performed by immunohistochemistry against the astroglial marker GFAP, a major component of intermediate filaments in astrocytes, in the different brain regions available which included the motor, sensory, and frontal cortices as well as the striatum in the basal ganglia.\u003c/p\u003e \u003cp\u003eIn the motor cortex of the control sample, GFAP immunoreactivity in the grey matter was mainly found in cell bodies and processes of subpial astrocytes forming the glia limitans and in interlaminar astrocytes of the cortical layer I (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,c). Subpial astrocytes showed intense GFAP labeling, and the cell bodies and proximal radial processes of interlaminar astrocytes showed medium-intensity GFAP staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). There were scarce GFAP\u0026thinsp;+\u0026thinsp;astrocytes with radial processes characteristic of protoplasmic astrocytes across the rest of the layers of the cortex, usually associated to microvessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee,g,i). Fibrous astrocytes were found in the subcortical white matter (WM), with small cell bodies and processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek). The intensity of GFAP expression in the subcortical WM was medium to low and similar between cell bodies and processes. The motor cortex of the MCT8-deficient subject showed a different distribution of GFAP\u0026thinsp;+\u0026thinsp;cells in comparison to the control subject. GFAP\u0026thinsp;+\u0026thinsp;staining in the glia limitans displayed less density of subpial astrocytes than in control samples, but a similar density of interlaminar astrocytes in layer I (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,d). In contrast to the control, GFAP\u0026thinsp;+\u0026thinsp;cells with astrocytic features similar to the ones present in layer I were present throughout all cortical layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef,h,j), particularly at the gyrus, and were less pronounced at the banks and the sulcus where stained astrocytes were almost exclusively associated to microvessels at layers II-VI. The MCT8-deficient subcortical WM presented a higher density of fibrous astrocytes in comparison to controls, with intensely stained cell bodies and shorter processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el). In conclusion, the MCT8-deficient subject presented fewer subpial astrocytes at the glial limitans, and an increased number of fibrous astrocytes across layers II-VI and the subcortical WM, where astrocyte morphology also differed from control samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilar findings were observed at the sensory and frontal cortices (\u003cb\u003eSupplemental Fig.\u0026nbsp;1, online Resource 1\u003c/b\u003e). Control sensory and frontal cortices showed subpial astrocytes with intense GFAP expression, interlaminar astrocytes in layer I with medium GFAP intensity staining in cell bodies and proximal radial processes; scarce GFAP\u0026thinsp;+\u0026thinsp;astrocytes across the rest of cortical layers; and low GFAP stained cell bodies and processes in the subcortical WM. As observed in the motor cortex, GFAP\u0026thinsp;+\u0026thinsp;cells in the MCT8-deficient sensory and frontal cortices were present throughout all cortical layers, mainly in the gyral convexity, and the density of fibrous astrocytes in the subcortical WM was higher than in control samples.\u003c/p\u003e \u003cp\u003eThe analysis of the GFAP\u0026thinsp;+\u0026thinsp;astrocytes in the basal ganglia was performed in the available tissue, which included the caudate and putamen of the striatum. Prominent GFAP immunolabeling was identified in the proximity of blood vessels both in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and MCT8-deficient (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) samples. Even though there were no apparent differences in the density of GFAP\u0026thinsp;+\u0026thinsp;cells, the staining intensity of GFAP\u0026thinsp;+\u0026thinsp;cells appeared to be higher in MCT8-deficient samples in comparison to the control subject (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to validate and further explore the astroglial alterations found in the human MCT8-deficient samples, we performed immunohistochemistry against two different astroglial markers, GFAP and ALDH1L1, in brain samples of a validated model of the AHDS: the \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mouse model (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Studies were initially performed in 3-month-old mice, an age that corresponds to an 11-year-old human (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), focusing on the motor cortex and the basal ganglia, as for the human samples.\u003c/p\u003e \u003cp\u003eIn WT animals, GFAP\u0026thinsp;+\u0026thinsp;astrocytes were present mainly in the upper layers of the motor cortex, with restricted GFAP signal to the soma and initial part of the astrocytic processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,c,e). In contrast, in the motor cortex of \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice astroglial cells were found throughout all cortical layers showing an increased cell density with higher GFAP signal in astroglial processes and cell bodies than in WT animals (2.41 fold-change increase; t(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;25.11, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,d,f,m). ALDH1L1 immunolabeling exhibited a broader expression pattern in WT samples in comparison to GFAP in the motor cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg,I,k), as previously described (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In the motor cortex, ALDH1L1 immunostaining was present in all cortical layers of WT and \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice, although the ALDH1L1\u0026thinsp;+\u0026thinsp;cell density was higher in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO samples (1.46 fold-change increase; t(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;9.085, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh,j,l,n). Notably, ALDH1L1 staining intensity in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice was higher in astroglial processes and cell bodies than in WT animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei-l).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the striatum of the basal ganglia, GFAP and ALDH1L1 immunolabeling displayed a similar density of GFAP+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b) and ALDH1L1+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee,f) cells in the WT and \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice, resembling the findings in human samples. Moreover, GFAP staining intensity appeared to be higher in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO than in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec,d), and no apparent differences in staining intensity were observed for ALDH1L1 immunolabeling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo discern the relative roles of DIO2 and MCT8 proteins in the immunostaining pattern of GFAP, \u003cem\u003eDio2\u003c/em\u003eKO, and \u003cem\u003eMct8\u003c/em\u003eKO animals were also analyzed in the motor cortex. This analysis confirmed the increase in the number of GFAP\u0026thinsp;+\u0026thinsp;astrocytes in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice in comparison to WT (2.41 fold-change increase; Tukey's multiple comparisons test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Notably, there were no differences in the number of GFAP\u0026thinsp;+\u0026thinsp;astrocytes between WT and \u003cem\u003eDio2\u003c/em\u003eKO mice (Tukey's multiple comparisons test, p\u0026thinsp;=\u0026thinsp;0.0835) and a small increase in the number of GFAP\u0026thinsp;+\u0026thinsp;astrocytes in \u003cem\u003eMct8\u003c/em\u003eKO mice in comparison to WT (1.25 fold-change increase; Tukey's multiple comparisons test, p\u0026thinsp;=\u0026thinsp;0.0101, \u003cb\u003eSupplementary Fig.\u0026nbsp;2, online Resource 1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo assess if these changes in the morphology and number of astroglial cells were persistent at later stages, immunolabeling against GFAP and ALDH1L1 was performed in 6-month-old WT and \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice. The outcomes resembled the findings in 3-month-old mice: there was an increased number of GFAP+ (3.21 fold-change increase; t(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;11.57, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and ALDH1L1+ (1.47 fold-change increase; t(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;8.88, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) astrocytes throughout all the cortical layers with increased GFAP and ALDH1L1 labeling in the soma and processes of the astrocytes in the motor cortex of \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice compared to WT (\u003cb\u003eSupplementary Fig.\u0026nbsp;3, online Resource 1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe increase in GFAP\u0026thinsp;+\u0026thinsp;cells across all cortical layers are indicative of persistent astroglial alterations, compatible with an astrogliosis-like phenotype, in the cerebral cortex of a mouse model of AHDS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMCT8 deficiency leads to astroglial developmental alterations\u003c/h2\u003e \u003cp\u003eTo assess whether astroglial alterations are present during early brain development or whether they are acquired at later developmental stages, we performed immunostaining against GFAP in the occipital cortex from a 30th gestational week MCT8-deficient fetus.\u003c/p\u003e \u003cp\u003eThe occipital cortex of the MCT8 deficient fetus showed poorly defined lamination, smaller thickness of cortical layers (in particular layers II, III, and IV), and higher cell density in comparison to a control subject (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,b), as previously described (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). In layer I of the occipital cortex, GFAP immunostaining was altered in the MCT8-deficient fetus with decreased, or even absent, GFAP\u0026thinsp;+\u0026thinsp;signal at cell bodies and processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Layers II-IV did not present distinctive GFAP labeling either in the control or the MCT8-deficient patient (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee-h). Layers V and VI presented GFAP\u0026thinsp;+\u0026thinsp;radial glia processes both in the control and the MCT8-deficient fetus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei,j). Striking differences were observed in the transition between layer VI and the subcortical WM and in subcortical WM itself, where the control sample displayed well-organized radial glial fibers with highly polarized astrocytes, while the MCT8-deficient fetus presented less GFAP\u0026thinsp;+\u0026thinsp;radial glia fibers and less polarized processes emerging from GFAP\u0026thinsp;+\u0026thinsp;astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek,l). These findings suggest that the astrogliosis-like phenotype observed in the 11-year-old MCT8-deficient subject is not present during fetal brain development, however, there seem to be defects on radial glia fibers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to validate and further explore the astroglial alterations found in human MCT8-deficient samples during brain development, we performed immunohistochemistry against GFAP in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mouse occipital cortices, in particular, in the visual cortex. We studied mice at postnatal day 7 (P7), the stage of early differentiation of astrocytes (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), and at P15, which corresponds to the period of late differentiation and maturation of astrocytes, equivalent to a 30th gestational week human fetus (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimilar to the findings observed in the MCT8-deficient human fetal occipital cortex, Nissl staining of the visual cortex revealed that the cytoarchitecture of the cortical layers is disorganized in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO animals in comparison to WT, both at P7 and P15. At P7, the thickness of the cortical layers (in particular layers II-IV) of \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice was decreased in comparison to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea,b). \u003cem\u003eMct8/Dio2\u003c/em\u003eKO also displayed higher cell density in the upper layers, lower cell density in the infragranular layers, and poorly defined boundaries between cortical layers compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea,b). At P15, \u003cem\u003eMct8/Dio2\u003c/em\u003eKO animals presented poorly defined boundaries between cortical layers in comparison to WT mice, however, there were no apparent differences in the cell density between the two genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec,d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of the distribution of astrocytes in the different layers of the visual cortex of WT and \u003cem\u003eMct8/Dio2\u003c/em\u003eKO animals at P7 showed a very similar distribution of GFAP\u0026thinsp;+\u0026thinsp;cells in both genotypes, with immunopositive cells located mainly in layer I (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee,f). However, \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice presented a decrease in immunolabeled GFAP\u0026thinsp;+\u0026thinsp;fibers, most likely radial glia processes, in comparison to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee-h). At P15, radial glia processes were already absent in both WT and \u003cem\u003eMct8/Dio2\u003c/em\u003eKO animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei-l). At this age, GFAP\u0026thinsp;+\u0026thinsp;astrocytes in WT animals concentrated mainly in layers I, II, and VI (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei,k), while in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO animals there was an increase in GFAP\u0026thinsp;+\u0026thinsp;astrocytes distributed throughout all cortical layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ej,l). In addition, GFAP\u0026thinsp;+\u0026thinsp;astrocytes presented greater intensity of GFAP labeling, as well as an increased number of processes and ramifications in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice in comparison to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei-l). These findings validate the observations in the human samples indicating that the astrogliosis-like phenotype observed in adult \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice is not present during early brain development, and that it develops between P7 and P15. Additionally, findings in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice support defects in the radial glia.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eGiven the importance of THs action during astrocyte development, as well as recent evidence suggesting that hypothyroidism might lead to gliosis (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), in this work we have evaluated possible alterations in the astroglial population including a potential state of astrogliosis as an additional pathological mechanism mediating the severe neurological impairments in MCT8-deficient patients.\u003c/p\u003e \u003cp\u003eWe have analyzed MR images to identify \u003cem\u003ein vivo\u003c/em\u003e alterations in the brain cytoarchitecture of MCT8-deficient patients, which may be a predictor of gliosis and tissular damage. Analysis of brain water diffusivity by ADC, frequently seen with gliosis in different cerebral pathologies such as adrenoleukodystrophies or Alzheimer\u0026rsquo;s disease (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), revealed increases in ADC values in the cerebral cortex and the striatum of MCT8-deficient patients in comparison to controls, indicating brain microstructure alterations. Increases in ADC values might also be associated with potential gliosis in MCT8 deficiency. Indeed, we have identified important alterations in the number and distribution of astroglial cell populations in the motor, sensory, and frontal cortices from an 11-year-old human MCT8-deficient subject. These included increased number of astrocytes particularly across layers III-VI and in the subcortical WM, suggestive of reactive astrogliosis in MCT8 deficiency. However, no apparent changes in the number of astrocytes were observed in the basal ganglia of the MCT8-deficient subject or in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice. Increases in ADC values in the striatum of MCT8-deficient patients, in combination with previously identified changes in fractional anisotropy by MRI in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice [13], may be indicative of axonal degeneration in the striatum [28].\u003c/p\u003e \u003cp\u003eThis observation was validated in a mouse model of MCT8 deficiency at 3 months of age, the correlating age to an 11-year-old human (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). We found an increased density of GFAP\u0026thinsp;+\u0026thinsp;and ALDH1L1\u0026thinsp;+\u0026thinsp;cells in the cerebral cortex of \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice in comparison to control samples, confirming the existence of astroglial alterations, compatible with astrogliosis, associated with MCT8 deficiency at preadolescent stages. In addition, the presence of alterations in GFAP\u0026thinsp;+\u0026thinsp;immunostaining in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice at 6 months of age is suggestive of persistent astroglial alterations in MCT8 deficiency at later stages.\u003c/p\u003e \u003cp\u003eMoreover, the mild alterations in GFAP immunostaining in single \u003cem\u003eMct8\u003c/em\u003eKO mice indicate that astroglial alterations in MCT8 deficiency are likely due to the state of brain hypothyroidism (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) arising from impaired THs transport into the brain across the brain barriers (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), rather than to the lack of MCT8 in the astrocytes. In the absence of MCT8, THs transport into astrocytes could be compensated by the presence of additional THs transporters, such as LAT1 (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Indeed, there is increasing evidence indicating that brain hypothyroidism leads to astrogliosis. It has been observed that hypothyroidism induced by anti-thyroid drugs increases the number of GFAP\u0026thinsp;+\u0026thinsp;cells in the cortex (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and hippocampus (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) and that brain-hypothyroid-mice deficient in MCT8 and the organic anion transporting protein OATP1C1 present an elevated number of astrocytes in the corpus callosum (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Our findings further support this emerging body of literature linking hypothyroidism to astrogliosis and extend these observations to humans. Such revelations could have an important impact beyond the MCT8 deficiency field in other hypothyroid conditions such as, for example, those derived from congenital hypothyroidism, Hashimoto's disease, thyroiditis, low iodine diet, or endocrine disruptors.\u003c/p\u003e \u003cp\u003eFurther studies are necessary to determine if the astroglial alterations observed in the cerebral cortex are a result of the brain insults derived from MCT8 deficiency, due to alterations in the development of the astroglial lineage, or both. Here we have observed that brain samples from a 30th gestational week MCT8-deficient fetus presented less GFAP\u0026thinsp;+\u0026thinsp;cells only in layer I of the occipital cortex and no changes in the number of astrocytes were observed in the visual cortex of \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice at P7. However, by P15 there was an evident increase in the number of astrocytes in the cortex of \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice. On one hand, this could suggest that the increase in GFAP\u0026thinsp;+\u0026thinsp;cells is a response to early brain insults that take place between P7 and P15. On the other hand, this could point to alterations arising during the generation of astrocytes. One of the main sources of astrocytes are neural stem cells (NSCs) that, following the \u0026ldquo;gliogenic switch\u0026rdquo;, stop generating neurons and predominantly generate oligodendrocyte and astrocyte lineage cells (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). The differentiation of NSCs into specific glial populations is determined by the sustained expression of a single basic helix-loop-helix transcription factor, in particular, \u003cem\u003eOlig2\u003c/em\u003e expression will give rise to oligodendrocyte precursor cells (OPCs) and \u003cem\u003eHes1\u003c/em\u003e to astrocytes (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Interestingly, \u003cem\u003eOlig2\u003c/em\u003e is a positively T3-regulated gene (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) and T3 has been shown to negatively regulate other basic helix-loop-helix transcription factors from the Hes gene family such as \u003cem\u003eHes7\u003c/em\u003e and \u003cem\u003eHes5\u003c/em\u003e (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). This suggests that the brain hypothyroid state in MCT8 deficiency might be promoting the generation of astrocytes from NSCs which could be the underlying cause for the elevated number of astrocytes associated with MCT8 deficiency.\u003c/p\u003e \u003cp\u003eRegardless of the nature of the increase in the number of astrocytes at P15 in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO mice, these alterations in the astroglial population could be severely impacting other neurodevelopmental processes. For example, synaptic pruning, a synaptic maturation process with a synapse elimination phase, is primarily mediated by astroglial cells (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). This illustrates how alterations in astroglia could lead to other defects, therefore affecting the refinement of neuronal circuits during development.\u003c/p\u003e \u003cp\u003eFinally, we have also observed less GFAP\u0026thinsp;+\u0026thinsp;radial glia processes in the MCT8-deficient fetus in comparison to controls. This finding was corroborated in \u003cem\u003eMct8/Dio2\u003c/em\u003eKO animals at P7 that also presented fewer radial glia processes than WT in the visual cortex. This is of relevance as we have previously identified the radial glia as an important regulator of THs availability to the human developing brain, as it presents the TH transporters MCT8 and OATP1C1 as well as DIO2 and the enzyme deiodinase 3 (DIO3) (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). This suggests that impaired radial glia development in MCT8 deficiency may impact the local availability of THs to neural cells that are dependent on this THs availability pathway. The alterations in radial glia processes here identified are likely mediating the cytoarchitecture alterations observed both in the MCT8-deficient human fetus and \u003cem\u003eMct8/Dio2\u003c/em\u003eKO animals at P7 and P15, which include higher cell density, decreased thickness of cortical layers, and poorly defined lamination. This could be because, during corticogenesis, newly generated neuroblasts use radial glia fibers as a scaffold for migration towards the upper cortical layers (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Nevertheless, the observed reduction in cortical thickness could also be due to defects in proliferation and a decreased number of neural progenitors in hypothyroid conditions, as previously described (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eBased on the current findings we conclude that MCT8 deficiency leads to alterations in astroglia, consistent with astrogliosis, from early stages of development that are persistent at adult stages. Impaired T3 signaling in NSCs could be a contributing factor to the elevated astrocyte number and changes in astrocyte morphology. Defects in radial glia might be underlying the alterations in brain cytoarchitecture identified by ADC and histochemistry in MCT8-deficient patients. Moreover, our findings point to ADC analysis as a possible \u003cem\u003ein vivo\u003c/em\u003e non-invasive marker of cytoarchitecture alterations in MCT8-deficiency and might be a predictor of astroglia imbalance to evaluate the progression of neurological impairments in patients, as well as the outcome of potential therapeutic strategies. In summary, MCT8 deficiency leads to an astrogliosis-like phenotype that arises as a novel pathological mechanism underlying the severe neurological defects present in patients. In view of this, astrocytes appear as new therapeutic targets for AHDS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe processing and use of the human tissue samples were approved by the ethics committee of Consejo Superior de Investigaciones Científicas (CSIC; numbers of permission: 045/2019 for SAF2017-86342-R and 124/2022 for PID2020-113139RB-I00). Human samples and data were obtained with informed consent from the families, in agreement with the Declaration of Helsinki. Terms and conditions to analyze the human brain MRI images were approved by an Authorized Official of both Institutions: The Medical University of South Carolina and Instituto de Investigaciones Biomédicas Sols-Morreale by means of a Data Transfer and Use Agreement. In all cases, personal data were treated anonymously.\u003c/p\u003e\n\u003cp\u003eAll experimental procedures involving animals were performed following the European Union Council guidelines (directive 2010/63/UE) and Spanish regulations (R.D. 53/2013) and were approved by the ethics committee Comité de Ética y Experimentación Humana y Animal (CEEHA) and by the Comunidad Autónoma de Madrid Review Board for the use of animals for scientific purposes (approval numbers 162/17, 252.7/20 and 014.1/21).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article and its additional files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are extremely grateful to the patients’ parents who gave their consent to use the brain tissues and the MR images for this investigation. We are also indebted to the IdiPAZ Biobank (PT20/00004) and the Sant Joan de Déu Hospital Biobank integrated into the Spanish National Biobanks and the Sydney Children’s and Prince of Wales Hospitals, Randwick, Australia as well as the Wolfson Medical Center,\u0026nbsp;Holon, Israel, and the Sackler School of Medicine, Tel Aviv, Israel, for the generous gifts of clinical samples used in this work. We thank Daniela López-Espíndola for her contribution to the fetal brain initial studies and Estrella Rausell, Laura Barrios and Belén Garzón for their helpful advice. We would like to thank María Camino de Lucas, Cristina Crespo Fernández, and Coral Pedrero García for animal care.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by 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 (Grant No. 2020AEP044 to AG-F), The Sherman Foundation (Grant No.\u0026nbsp;OTR02211 to SB-L and AG-F), Asociación Corriendo con el Corazón por Hugo (Grant No. OTR06190 to AG-F), a contract from Ministerio de Ciencia, Innovación y Universidades (Programa de Formación de Profesorado (FPU, FPU19/02006) to MG-Y, a contract from Universidad Autónoma de Madrid (Contrato predoctoral para Formación de personal Investigador en Formación, FPI-UAM) to AG-A, a contract from MCIN/AEI/10.13039/501100011033 and “ESF Investing in your future” (Grant No.\u0026nbsp;PRE2018-086185) to VV-H, and a grant from the MCIN/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTR (Grant No. IJC2020-043543-I) to SB-L.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors and affiliations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLaboratory of Thyroid hormones and CNS. Department of Neurological Diseases and Aging.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eInstituto de Investigaciones Biomédicas Sols-Morreale, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), Madrid, Spain.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMarina Guillén-Yunta, Ángel García-Aldea, Víctor Valcárcel-Hernández, Ainara Sanz-Bógalo, Carmen Grijota-Martínez, Soledad Bárez-López, Ana Montero-Pedrazuela \u0026amp; Ana Guadaño-Ferraz.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMagnetic Imaging Resonance Core Facility, Institut d'Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), Barcelona, Spain.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEmma Muñoz-Moreno.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Radiology and Radiological Science, Medical University of South Carolina, Charleston, SC, USA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaria Gisele Matheus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Cell Biology, Faculty of Biology, Universidad Complutense de Madrid, Madrid, Spain.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCarmen Grijota-Martínez\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors’ contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMG-Y, AG-A, CG-M, SB-L, AM-P and AG-F conceived and planed the experimental procedures. MG-Y, ÁG-A, VV-H, AS-B, CG-M, SB-L and AM-P carried out the experiments and took care of the colony at our animal facility. MG-Y, SB-L and AM-P quantified and analyzed data obtained from the experimental procedures. MG-M provided the MRI images from control and MCT8-deficient subjects and EM-M segmented and quantified the ADC values. AG-F supervised and administered the research project. SB-L wrote the original manuscript with the support from MG-Y, AM-P and AG-F. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAllan W, Herndon C, Dudley FC. Some examples of the inheritance of mental deficiency: apparently sex-linked idiocy and microcephaly. Am J Ment Defic. 1944;48(48):325-34.\u003c/li\u003e\n\u003cli\u003eDumitrescu AM, Liao X-H, Best TB, Brockmann K, Refetoff S. A Novel Syndrome Combining Thyroid and Neurological Abnormalities Is Associated with Mutations in a Monocarboxylate Transporter Gene. The American Journal of Human Genetics. 2004;74(1):168-75.\u003c/li\u003e\n\u003cli\u003eFriesema ECH, Grueters A, Biebermann H, Krude H, von Moers A, Reeser M, et al. Association between mutations in a thyroid hormone transporter and severe X-linked psychomotor retardation. The Lancet. 2004;364(9443):1435-7.\u003c/li\u003e\n\u003cli\u003eFriesema EC, Ganguly S, Abdalla A, Manning Fox JE, Halestrap AP, Visser TJ. Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter. J Biol Chem. 2003;278(41):40128-35.\u003c/li\u003e\n\u003cli\u003eMasnada S, Sarret C, Antonello CE, Fadilah A, Krude H, Mura E, et al. Movement disorders in MCT8 deficiency/Allan-Herndon-Dudley Syndrome. Mol Genet Metab. 2022;135(1):109-13.\u003c/li\u003e\n\u003cli\u003eRemerand G, Boespflug-Tanguy O, Tonduti D, Touraine R, Rodriguez D, Curie A, et al. Expanding the phenotypic spectrum of Allan\u0026ndash;Herndon\u0026ndash;Dudley syndrome in patients with SLC16A2 mutations. Developmental Medicine \u0026amp; Child Neurology. 2019;61(12):1439-47.\u003c/li\u003e\n\u003cli\u003eMatheus MG, Lehman RK, Bonilha L, Holden KR. Redefining the Pediatric Phenotype of X-Linked Monocarboxylate Transporter 8 (MCT8) Deficiency: Implications for Diagnosis and Therapies. J Child Neurol. 2015;30(12):1664-8.\u003c/li\u003e\n\u003cli\u003eCeballos A, Belinchon MM, Sanchez-Mendoza E, Grijota-Martinez C, Dumitrescu AM, Refetoff S, et al. Importance of monocarboxylate transporter 8 for the blood-brain barrier-dependent availability of 3,5,3\u0026apos;-triiodo-L-thyronine. Endocrinology. 2009;150(5):2491-6.\u003c/li\u003e\n\u003cli\u003eL\u0026oacute;pez-Esp\u0026iacute;ndola D, Garc\u0026iacute;a-Aldea \u0026Aacute;, G\u0026oacute;mez de la Riva I, Rodr\u0026iacute;guez-Garc\u0026iacute;a AM, Salvatore D, Visser TJ, et al. Thyroid hormone availability in the human fetal brain: novel entry pathways and role of radial glia. Brain Struct Funct. 2019;224(6):2103-19.\u003c/li\u003e\n\u003cli\u003eRoberts LM, Woodford K, Zhou M, Black DS, Haggerty JE, Tate EH, et al. Expression of the thyroid hormone transporters monocarboxylate transporter-8 (SLC16A2) and organic ion transporter-14 (SLCO1C1) at the blood-brain barrier. Endocrinology. 2008;149(12):6251-61.\u003c/li\u003e\n\u003cli\u003eL\u0026oacute;pez-Esp\u0026iacute;ndola D, Morales-Bastos C, Grijota-Mart\u0026iacute;nez C, Liao XH, Lev D, Sugo E, et al. Mutations of the thyroid hormone transporter MCT8 cause prenatal brain damage and persistent hypomyelination. J Clin Endocrinol Metab. 2014;99(12):E2799-804.\u003c/li\u003e\n\u003cli\u003eVancamp P, Demeneix BA, Remaud S. Monocarboxylate Transporter 8 Deficiency: Delayed or Permanent Hypomyelination? Front Endocrinol (Lausanne). 2020;11:283.\u003c/li\u003e\n\u003cli\u003eValc\u0026aacute;rcel-Hern\u0026aacute;ndez V, L\u0026oacute;pez-Esp\u0026iacute;ndola D, Guill\u0026eacute;n-Yunta M, Garc\u0026iacute;a-Aldea \u0026Aacute;, L\u0026oacute;pez de Toledo Soler I, B\u0026aacute;rez-L\u0026oacute;pez S, et al. Deficient thyroid hormone transport to the brain leads to impairments in axonal caliber and oligodendroglial development. Neurobiol Dis. 2022;162:105567.\u003c/li\u003e\n\u003cli\u003eGuill\u0026eacute;n-Yunta M, Valc\u0026aacute;rcel-Hern\u0026aacute;ndez V, Garc\u0026iacute;a-Aldea \u0026Aacute;, Soria G, Garc\u0026iacute;a-Verdugo JM, Montero-Pedrazuela A, et al. Neurovascular unit disruption and blood-brain barrier leakage in MCT8 deficiency. Fluids Barriers CNS. 2023;20(1):79.\u003c/li\u003e\n\u003cli\u003eManzano J, Bernal J, Morte B. Influence of thyroid hormones on maturation of rat cerebellar astrocytes. Int J Dev Neurosci. 2007;25(3):171-9.\u003c/li\u003e\n\u003cli\u003eDezonne RS, Stipursky J, Gomes FC. Effect of thyroid hormone depletion on cultured murine cerebral cortex astrocytes. Neurosci Lett. 2009;467(2):58-62.\u003c/li\u003e\n\u003cli\u003eMorte B, Gil-Ib\u0026aacute;\u0026ntilde;ez P, Bernal J. Regulation of Gene Expression by Thyroid Hormone in Primary Astrocytes: Factors Influencing the Genomic Response. Endocrinology. 2018;159(5):2083-92.\u003c/li\u003e\n\u003cli\u003eBernal J, Morte B, Diez D. Thyroid hormone regulators in human cerebral cortex development. J Endocrinol. 2022;255(3):R27-r36.\u003c/li\u003e\n\u003cli\u003eWang T, Wang Y, Montero-Pedrazuela A, Prensa L, Guada\u0026ntilde;o-Ferraz A, Rausell E. Thyroid Hormone Transporters MCT8 and OATP1C1 Are Expressed in Projection Neurons and Interneurons of Basal Ganglia and Motor Thalamus in the Adult Human and Macaque Brains. Int J Mol Sci. 2023;24(11).\u003c/li\u003e\n\u003cli\u003eBernal J, Guada\u0026ntilde;o-Ferraz A, Morte B. Thyroid hormone transporters--functions and clinical implications. Nat Rev Endocrinol. 2015;11(7):406-17.\u003c/li\u003e\n\u003cli\u003eGuada\u0026ntilde;o-Ferraz A, Obreg\u0026oacute;n MJ, St Germain DL, Bernal J. The type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain. Proc Natl Acad Sci U S A. 1997;94(19):10391-6.\u003c/li\u003e\n\u003cli\u003eNiedowicz DM, Wang WX, Price DA, Xie K, Patel E, Nelson PT. Impact of thyroid hormone perturbations in adult mice: brain weight and blood vessel changes, gene expression variation, and neurobehavioral outcomes. Neurobiol Aging. 2023;128:74-84.\u003c/li\u003e\n\u003cli\u003eMayerl S, Heuer H. Thyroid hormone transporter Mct8/Oatp1c1 deficiency compromises proper oligodendrocyte maturation in the mouse CNS. Neurobiol Dis. 2023;184:106195.\u003c/li\u003e\n\u003cli\u003eChamas L, Seugnet I, Poirier R, Clerget-Froidevaux MS, Enderlin V. A Fine Regulation of the Hippocampal Thyroid Signalling Protects Hypothyroid Mice against Glial Cell Activation. Int J Mol Sci. 2022;23(19).\u003c/li\u003e\n\u003cli\u003eMatusova Z, Hol EM, Pekny M, Kubista M, Valihrach L. Reactive astrogliosis in the era of single-cell transcriptomics. Front Cell Neurosci. 2023;17:1173200.\u003c/li\u003e\n\u003cli\u003eMu\u0026ntilde;oz-Ballester C, Robel S. Astrocyte-mediated mechanisms contribute to traumatic brain injury pathology. WIREs Mech Dis. 2023:e1622.\u003c/li\u003e\n\u003cli\u003eSofroniew MV. Astrogliosis. Cold Spring Harb Perspect Biol. 2014;7(2):a020420.\u003c/li\u003e\n\u003cli\u003eVoorn JPvd, Pouwels PJW, Powers JM, Kamphorst W, Martin J-J, Troost D, et al. Correlating Quantitative MR Imaging with Histopathology in X-Linked Adrenoleukodystrophy. American Journal of Neuroradiology. 2011;32(3):481-9.\u003c/li\u003e\n\u003cli\u003eLodygensky GA, West T, Stump M, Holtzman DM, Inder TE, Neil JJ. In vivo MRI analysis of an inflammatory injury in the developing brain. Brain, Behavior, and Immunity. 2010;24(5):759-67.\u003c/li\u003e\n\u003cli\u003eB\u0026aacute;rez-L\u0026oacute;pez S, Grijota-Mart\u0026iacute;nez C, Aus\u0026oacute; E, Fern\u0026aacute;ndez-de Frutos M, Montero-Pedrazuela A, Guada\u0026ntilde;o-Ferraz A. Adult Mice Lacking Mct8 and Dio2 Proteins Present Alterations in Peripheral Thyroid Hormone Levels and Severe Brain and Motor Skill Impairments. Thyroid. 2019;29(11):1669-82.\u003c/li\u003e\n\u003cli\u003eValc\u0026aacute;rcel-Hern\u0026aacute;ndez V, Guill\u0026eacute;n-Yunta M, Bueno-Arribas M, Montero-Pedrazuela A, Grijota-Mart\u0026iacute;nez C, Markossian S, et al. A CRISPR/Cas9-engineered avatar mouse model of monocarboxylate transporter 8 deficiency displays distinct neurological alterations. Neurobiol Dis. 2022;174:105896.\u003c/li\u003e\n\u003cli\u003eAvants BB, Epstein CL, Grossman M, Gee JC. Symmetric diffeomorphic image registration with cross-correlation: evaluating automated labeling of elderly and neurodegenerative brain. Med Image Anal. 2008;12(1):26-41.\u003c/li\u003e\n\u003cli\u003eFischl B, Salat DH, Busa E, Albert M, Dieterich M, Haselgrove C, et al. Whole brain segmentation: automated labeling of neuroanatomical structures in the human brain. Neuron. 2002;33(3):341-55.\u003c/li\u003e\n\u003cli\u003eDumitrescu AM, Liao XH, Weiss RE, Millen K, Refetoff S. Tissue-specific thyroid hormone deprivation and excess in monocarboxylate transporter (mct) 8-deficient mice. Endocrinology. 2006;147(9):4036-43.\u003c/li\u003e\n\u003cli\u003eSchneider MJ, Fiering SN, Pallud SE, Parlow AF, St Germain DL, Galton VA. Targeted disruption of the type 2 selenodeiodinase gene (DIO2) results in a phenotype of pituitary resistance to T4. Mol Endocrinol. 2001;15(12):2137-48.\u003c/li\u003e\n\u003cli\u003eDutta S, Sengupta P. Men and mice: Relating their ages. Life Sci. 2016;152:244-8.\u003c/li\u003e\n\u003cli\u003eMorel L, Men Y, Chiang MSR, Tian Y, Jin S, Yelick J, et al. Intracortical astrocyte subpopulations defined by astrocyte reporter Mice in the adult brain. Glia. 2019;67(1):171-81.\u003c/li\u003e\n\u003cli\u003eAkdemir ES, Huang AY, Deneen B. Astrocytogenesis: where, when, and how. F1000Res. 2020;9.\u003c/li\u003e\n\u003cli\u003eLattke M, Guillemot F. Understanding astrocyte differentiation: Clinical relevance, technical challenges, and new opportunities in the omics era. WIREs Mech Dis. 2022;14(5):e1557.\u003c/li\u003e\n\u003cli\u003eKantarci K, Petersen RC, Boeve BF, Knopman DS, Weigand SD, O\u0026apos;Brien PC, et al. DWI predicts future progression to Alzheimer disease in amnestic mild cognitive impairment. Neurology. 2005;64(5):902-4.\u003c/li\u003e\n\u003cli\u003eIwayama H, Liao XH, Braun L, B\u0026aacute;rez-L\u0026oacute;pez S, Kaspar B, Weiss RE, et al. Adeno Associated Virus 9-Based Gene Therapy Delivers a Functional Monocarboxylate Transporter 8, Improving Thyroid Hormone Availability to the Brain of Mct8-Deficient Mice. Thyroid. 2016;26(9):1311-9.\u003c/li\u003e\n\u003cli\u003eVatine GD, Al-Ahmad A, Barriga BK, Svendsen S, Salim A, Garcia L, et al. Modeling Psychomotor Retardation using iPSCs from MCT8-Deficient Patients Indicates a Prominent Role for the Blood-Brain Barrier. Cell Stem Cell. 2017;20(6):831-43.e5.\u003c/li\u003e\n\u003cli\u003eYamaguchi M, Seki T, Imayoshi I, Tamamaki N, Hayashi Y, Tatebayashi Y, et al. Neural stem cells and neuro/gliogenesis in the central nervous system: understanding the structural and functional plasticity of the developing, mature, and diseased brain. J Physiol Sci. 2016;66(3):197-206.\u003c/li\u003e\n\u003cli\u003eGil-Iba\u0026ntilde;ez P, Garc\u0026iacute;a-Garc\u0026iacute;a F, Dopazo J, Bernal J, Morte B. Global Transcriptome Analysis of Primary Cerebrocortical Cells: Identification of Genes Regulated by Triiodothyronine in Specific Cell Types. Cereb Cortex. 2017;27(1):706-17.\u003c/li\u003e\n\u003cli\u003ePerez-Catalan NA, Doe CQ, Ackerman SD. The role of astrocyte-mediated plasticity in neural circuit development and function. Neural Dev. 2021;16(1):1.\u003c/li\u003e\n\u003cli\u003eRakic P. Mode of cell migration to the superficial layers of fetal monkey neocortex. J Comp Neurol. 1972;145(1):61-83.\u003c/li\u003e\n\u003cli\u003eMohan V, Sinha RA, Pathak A, Rastogi L, Kumar P, Pal A, et al. Maternal thyroid hormone deficiency affects the fetal neocorticogenesis by reducing the proliferating pool, rate of neurogenesis and indirect neurogenesis. Exp Neurol. 2012;237(2):477-88.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Thyroid hormones, Thyroid hormone transporters, MCT8, Astroglia, MCT8 deficiency, MRI.","lastPublishedDoi":"10.21203/rs.3.rs-3825537/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3825537/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Allan-Herndon-Dudley syndrome (AHDS) is a rare X-linked disorder that causes severe neurological damage, for which there is no effective treatment. AHDS is due to inactivating mutations in the thyroid hormone transporter MCT8 that impair the entry of thyroid hormones into the brain, resulting in cerebral hypothyroidism. However, the pathophysiology of AHDS is still not fully understood and this is essential to develop therapeutic strategies. Based on evidence suggesting that thyroid hormone deficit leads to alterations in astroglial cells, including gliosis, in this work we have evaluated astroglial impairments in MCT8 deficiency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: We conducted magnetic resonance imaging on both control subjects and MCT8-deficient patients to examine changes in brain cytoarchitecture. Moreover, to gain further understanding on these alterations in brain cytoarchitecture and the astroglial population, we have performed histological and immunohistochemical approaches in autopsy brain samples from an 11-year-old and a 30\u003csup\u003eth\u003c/sup\u003e gestational week MCT8-deficient subjects in comparison to brain samples from control subjects at similar ages. Findings from MCT8-deficient subjects were validated and further explored in a mouse model of the AHDS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Magnetic resonance imaging showed changes indicative of alterations in brain cytoarchitecture in MCT8-deficient patients. Further studies confirmed changes in the astroglial population in MCT8 deficiency that arise early in brain development and persist at adult stages, revealing an abnormal distribution, density, and morphology of cortical astrocytes, compatible with an astrogliosis-like phenotype at adult stages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: We have identified astrocytes as potential novel therapeutic targets in AHDS. In addition, we propose ADC imaging as a tool to monitor the progression of neurological impairments and potential effects of treatments in MCT8 deficiency.\u003c/p\u003e","manuscriptTitle":"Defective thyroid hormone transport to the brain leads to astroglial alterations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-31 15:16:39","doi":"10.21203/rs.3.rs-3825537/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"25dceb60-ace5-4cd2-9e6d-dccacc662408","owner":[],"postedDate":"January 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-07T23:00:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-31 15:16:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3825537","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3825537","identity":"rs-3825537","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}