Impaired T3 uptake and action in MCT8-deficient cerebral organoids underlie the Allan-Herndon-Dudley syndrome

Patients with mutations in the thyroid hormone (TH) cell transporter MCT8 gene develop severe neuro-psychomotor retardation known as the Allan-Herndon-Dudley syndrome (AHDS). It is assumed that this is caused by a reduction in TH signaling in the developing brain, and treatment remains understandably challenging. Given species differences in brain TH transporters and the limitations of studies in mice, we generated brain organoids (BOs) using human iPSCs from MCT8-deficient patients. We found that MCT8-deficient BOs exhibit (i) impaired T3 transport in developing neural cells, as assessed through deiodinase-3-mediated T3 catabolism, (ii) reduced expression of genes involved in neurogenesis and neuronal maturation, and (iii) reduced T3-inducibility of TH-regulated genes. In contrast, the TH-analogs 3,5-diiodothyropropionic acid and 3,3’,5-triiodothyroacetic acid triggered normal responses (induction/repression of T3-responsive genes) in MCT8-deficient BOs, constituting a proof-of-concept that lack of T3 transport underlies the pathophysiology of AHDS, demonstrating the clinical potential for TH analogues to be used in treating AHDS patients. MCT8-deficient BOs represent a species-specific relevant preclinical model that can be utilized to screen drugs with potential benefits as personalized therapeutics for AHDS patients.


Introduction
Thyroid hormones (THs) are crucial for brain development and greatly influence brain function throughout life (1)(2)(3)(4)(5).TH relies on specific cell membrane transporters to enter the brain and neural cells, including the monocarboxylate transporter 8 (MCT8; encoded by SLC16A2 in the X chromosome) (6).MCT8 plays a critical role in TH signaling, as showcased by the profound phenotype observed in boys carrying loss-of-function mutations in SLC16A2, which is indicative of brain hypothyroidism during critical developmental stages.Patients with the Allan-Herndon-Dudley syndrome (a.k.a.AHDS) exhibit characteristic serum TH abnormalities [high triiodothyronine (T3), low thyroxine (T4), and reverse T3 (rT3), with normal or slightly elevated thyrotropin (TSH)] accompanied by severe and irreversible neurological deficits (7), presumably due to reduced TH availability to neural cells.This assumption is mainly circumstantial but also derived from a study that identified a ~50% reduction in TH contents in the cerebral cortex and abnormalities in neuronal differentiation, synaptogenesis, and myelination in brain sections of a fetus with AHDS (8,9).There are also MRI studies indicating hypomyelination during the first years of life (8,9), but it is unclear whether it persists into adulthood (10).
To gain greater insight into the pathophysiology of the AHDS, researchers have looked into the brain of animal models expressing a non-functional MCT8 and also studied neural cells derived from induced pluripotent stem cells (iPSCs) and found that MCT8 plays a role in the passage of TH through the blood-brain barrier (BBB) (11)(12)(13).Hence, the concept that MCT8 mediates TH transport into the brain parenchyma is well accepted.Notwithstanding, the fact that MCT8 is widely expressed in the human brain (13)(14)(15)(16) supports a broader role for MCT8 in T3 transport into neural cells.This seems to be the case in mouse neurons where Mct8 is critical in an endosomal pathway that retrogradely transports T3 from distal axons to the cell nucleus of these cells (17).Another example is the neural progenitor cells of chicken embryos and adult mouse hippocampi, where Mct8 functions as a gatekeeper in the process of neurogenesis (18,19).However, a unifying hypothesis for the contribution of MCT8 to TH transport and signaling in human neural cells and how its loss-of-function mutations relate to the neurological manifestations seen in patients is missing.
The treatment of patients with MCT8-deficiency remains challenging (20), and daily care poses a heavy burden on caregivers (21).Current options can only ameliorate thyroid test abnormalities associated with hypermetabolism without improvement of the severe neurologic defects.MCT8 deficiency cannot be corrected with high doses of TH due to its insufficient availability to the brain (22,23).Thus, studying TH analogs for their potential benefit in treating MCT8-deficient patients is paramount.In particular, the TH analogs 3,5-diiodothyropropionic acid (DITPA) and 3,3',5-triiodothyroacetic acid (TRIAC) do not depend on MCT8 for transport and have thyromimetic activity by binding to the TH receptors (24,25) and effect in Mct8deficient mice (26,27).DITPA also improves circulating T4 and T3 levels in MCT8-deficient humans (28) and promotes differentiation and myelination in healthy human oligodendrocytes (29).However, we still lack vital information about the role played by MCT8 and potential TH analogs in the human brain due to species differences in brain TH transporters.
To avoid these limitations, we generated cerebral organoids (COs) from patient-derived iPSCs and demonstrated that MCT8 is critical for early neurodevelopment, playing a role in the T3 uptake of developing human neural cells.The resulting impairment in transmembrane transport affects intracellular T3 action, including the expression of genes involved in cerebral cortex development and neural cell maturation.The present investigation reveals that DITPA and TRIAC can act in human neural cells bypassing a nonfunctional MCT8 and triggering responses similar to those induced by T3.These findings expand our understanding of the pathophysiology of AHDS and have broad implications for devising new potential treatments to improve the quality of life of patients with AHDS.

Generation of control and MCT8-deficient COs
We generated COs from four hiPSCs lines, two MCT8-deficient (Mut) and two healthy controls (WT): (i) Mut1, from a patient with a nonsense frameshift mutation A404fs416X (amino acids numbered according to the long MCT8 isoform), leading to a premature stop codon (ii) Mut2, from a patient with the missense mutation P321L, (iii) WT, control cell line from the father of Mut2 and (iv) isoWT, control isogenic line in which the mutation of Mut2 has been corrected by CRISPR/Cas9.(Supplemental Figure 1A, B).The four cell lines produced uniform round embryoid bodies (EBs) with smooth edges and diameters of about 500 μM and then developed to form optically translucent edges indicating neural induction (Figure 1A).During the next 10 days in culture, control and MCT8-deficient EBs (Figure 1A, B) showed neuroepithelial bud expansion and matured until they formed cerebral-like structures (Figure 1C), exhibiting similar growth (Figure 1D).At day 20 of culture (D20), control, and MCT8-deficient COs showed high expression of the forebrain markers FOXG1, NKX2-1, and LMX1B at significantly higher levels than the original hiPSCs (Figure 1E) while maintaining a high expression of the pluripotency markers OCT4 and ectoderm marker SOX2 (Figure 1F).

MCT8-CO exhibits impaired early neurogenesis
Control and MCT8-deficient D20 COs were able to initiate a normal neurodevelopmental pattern with multiple large and continuous small cortical units composed of a neural rosette constituting neuroepithelium-like structures similar to a ventricle (30).These neural rosettes are arranged in a laminar fashion, containing SOX2+ neural progenitors and Ki67+ proliferating cells (Figure 2A-C), resembling the ventricular zone (VZ) of the fetal cortex at about gestational week 6.5 (31).However, while WT and IsoWT COs formed neural rosettes with average diameters of 144.5 ± 28.5 µm containing 5.3 ± 1.1 layers of SOX2+ Ki67-cells, MCT8-COs formed smaller rosettes of 97.2 ± 26.4 µm with only 2.5 ± 1.0 layers of SOX2+ cells (Figure 2D, E).These alterations indicate a cell proliferation defect in the early stages of corticogenesis.To explore this hypothesis, we traced back the neural precursor cells, staining them for the mitotic marker phospho-histone H3 (PH3) and phospho-vimentin (PVIM; (32).In Control and MCT8-deficient COs, the majority of neural precursor cells were dividing at the apical surface, a typical feature of the neuroepithelium with apico-basal polarity (Figure 2F).We then measured their plane of nuclear migration (mitotic spindle) occurring during the translocation of the nuclei for mitosis and observed that Control COs exhibited mainly horizontal divisions (52.5 ± 5.6 %), which were less abundant in MCT8-deficient COs (40.4 ± 9.0 %, on average; Figure 2G).During human brain development, horizontal divisions contribute to normal neurogenesis by regulating cortical expansion (33,34).Thus the lower percentage of horizontal divisions in MCT8-deficient COs may explain the observed reductions in the diameter and thickness of the cortical units of the MCT8-deficient COs.
We next performed immunofluorescence labeling and gene expression studies of D20 COs to test for the presence of TUJ1+ postmitotic neurons and evaluate the expression of markers for neuronal differentiation and mature neurons.Control and MCT8-deficient COs were populated with TUJ1+ postmitotic neurons (Figure 2H).Furthermore, neurons exhibited a primitive separation with a late-born superficial layer featuring SATB2+ cells (inset in Figure 2H; (35)).
However, MCT8-deficient COs exhibited significantly reduced mRNA levels of the neuronal markers TUBB3 and NEUN and of the genes involved in neuronal differentiation JAG1 and HES5 (Figure 2I).

MCT8-deficient COs presented an altered expression of TH transporters
We first wanted to confirm that MCT8 was present in neurons residing in D20 WT COs.
Immunostaining for MCT8 and vimentin (a marker for neuronal cytoskeleton) revealed that MCT8 is evenly expressed in COs neurons (Figure 3A), including the TUJ1+ postmitotic neurons (Figure 3B).Next, we examined the mRNA levels of TH transporters.Compared to D20 Controls, MCT8-deficient COs exhibited a reduced expression of MCT8, MCT10, and LAT2 and a similar expression of LAT1 (Figure 3C).In these COs, we also measured the expression of the TH nuclear receptors and found that Control and MCT8-deficient COs exhibited a similar expression of THRA and THRB (Figure 3D).

MCT8-deficient COs exhibit reduced D3-mediated metabolism
Within the brain, the neurons express high levels of the TH-inactivating deiodinase-3 (D3) that metabolizes T3 to T2 (36,37), and glial cells express the activating enzyme D2, which metabolizes the prohormone, thyroxine (T4), to T3 (38)(39)(40).Here, we first studied changes in the DIO3 expression (encoding D3) and found no differences between groups (Figure 3E).Next, we measured D3 activity as a proxy to assess T3 transport into the neural cells.D20 COs were incubated for 24h with T3-I 125 , resulting in a prominent peak of T2-I 125 in the medium that was smaller in the MCT8-deficient COs (Figure 3F).Control COs exhibit a D3 activity of 18.4±7.7 pmol/mg/h, while the MCT8-deficient COs have ~30% of that in WT (5.3 ± 3.1 pmol/mg/h) (Figure 3G).To test whether the decrease in the D3-activity observed in the MCT8-deficient COs was mediated via a defective MCT8 transport, we next incubated the control COs with T3-I 125 in the presence of 2 µM of the highly selective MCT8 inhibitor Silychristin (WT+SC) (41), and indeed the D3 activity was decreased to 4.4 ± 3.0 pmol/mg/h (Figure 3G), a level similar to that observed in MCT8-deficient COs.Then, we studied changes in DIO2 expression (encoding D2) and found no differences between groups (Figure 3H).DIO2 expression peaked in D20 WT COs (Figure 3I), but we could not detect T3-I 125 production in the medium after incubating D20 COs for 24h with T4-I 125 .DIO2 is expressed in neural precursor cells (42)(43)(44), which also express the high affinity T3-binding cytoplasmic proteins μ-crystallin (CRYM (45)).Thus, it is conceivable that most of the T3-I 125 generated via D2 may remain in the cytosol trapped by this T3-binding cytoplasmatic protein.To explore this possibility further, we measured D2 activity in COs sonicates, which allowed us to identify D2-mediated T4-I 125 to T3-I 125 conversion of about 1.4 ± 0.7 pmol/mg/h in D15 COs, increasing to 3.9 ± 0.9 pmol/mg/h in D20 COs.Interestingly, these changes in D2 activity were paralleled by a ~89% increase in the expression of CRYM (Figure 3K).

DITPA and TRIAC can trigger TH signaling in MCT8-deficient COs
We next treated for 24h control and MCT8-deficient COs with either 10nM triiodothyronine (T3) and equivalent doses of DITPA (3.5µM), or TRIAC (10nM) and measured T3-responsive genes.
The respective changes in mRNA levels in the COs are shown in Figure 4.For genes upregulated by T3 in control COs: HAIRLESS and KLF9 (Figure 4A, B).T3 increased (P <0.05) the expression of HAIRLESS in Mut1 but did not affect Mut2 COs.Notably, T3 did not affect KLF9 mRNA levels in MCT8-deficient COs, but treatment with both DITPA and TRIAC increased (P <0.05) its expression in MCT8-deficient COs.For genes downregulated by T3 in control COs: CIRBP and COL6A1 (Figure 4C, D).T3 did not affect these two genes in Mut1 COs, but either DITPA or TRIAC reduced (P <0.01) the CIRBP and COL6A mRNA levels in these COs.In Mut2, however, T3 downregulated the expression of these genes, and the response was more significant when Mut2 COs were treated with TRIAC or DITPA.Overall, these results indicate that the T3-signalling in the MCT8-deficient COs might be compromised and represent a proof of concept that the thyromimetic molecules TRIAC and DITPA can act in human neural cells despite a non-functional MCT8.

Transcriptome analysis
To assess the effect of the T3 treatment (from D50 to D65) on the maturation of control and MCT8-deficient COs, we validated the presence of neurons, astrocytes, and oligodendrocytes in D65 COs by immunofluorescence imaging.D65 COs exhibited an evenly distributed population of NEUROD1+ differentiated neurons Figure 5B), GFAP+ cells with two different morphologies resembling glial precursor cells (Figure 5C), and astrocytes in culture co-expressing aquaporin 4 (AQP4; a water channel critical for astrocyte function; Figure 5D).In addition, D65 COs also contained abundant populations of OLIG2+ oligodendrocyte precursor cells and O4+ premyelinating oligodendrocytes (Figure 5E).In addition, we assessed the mRNA levels of OLIG2 (markers of oligodendrocyte precursor cells) and MBP (markers of myelinating oligodendrocytes).Analysis of D50 versus D65 COs showed that the OLIG2 mRNA levels increased similarly in WT (72.9 ± 25.7%) and Mut1 (68.5 ± 21.1%) and to a lesser extent in Mut2 COs (38.4 ± 12.9%).The levels of MBP mRNA increased at a similar rate (61.2 ± 10.8% on average) in both control and MCT8-deficient COs (Supplemental Figure 2A, B).
The discovery that MCT8 deficiency impairs T3 uptake and action in COs (Figures 3 and 4), combined with the fact that T3 is required to promote the maturation of COs, suggests a role for MCT8 in CO maturation.To explore the role of MCT8 in COs maturation, we next performed RNA-seq in D65 WT, Mut1, and Mut2 COs (after 15 days of treatment with 60nM T3; these datasets clustered separately in a principal component plot; Supplemental Figure 3A).
Compared to controls, Mut1 and Mut2 COs were associated with the differential expression of 1371 and 2969 genes, respectively (1228 and 1864 downregulated and 143 and 1105 upregulated; Figure 5F,G; Supplemental Tables 2 and 3).Among these genes, we identified a common cluster of 949 genes (cluster E; Figure 5H) that were either up-or downregulated (65 and 885, respectively, when comparing Control vs Mut1 and vs. Mut2 (Figure 5I; Supplemental Table 4 and 5).We then looked at the top 100 differentially expressed genes (DEGs) of cluster E and found that 63 were common for both MCT8-deficient COs (Figure 5J; Supplemental Table 6).Of these genes, the top gene SATB2 is known to be regulated by T3 (46) and encodes a transcription factor that regulates neuronal differentiation and specification, being reduced/absent in some individuals manifesting developmental delay, intellectual disability, and severe speech delay (47)-common alterations in patients with the AHDS.Another top gene, KNCF1, encodes a potassium ion channel involved in neurotransmitter release and neural excitation.Alterations in this gene have been associated with neurodevelopmental alterations in cerebral organoids (48) and epilepsy (49).The gene CRYM (also studied in Figure 3) was also found to be among the top downregulated genes in MCT8-deficient COs.The remaining genes included MMP2, a modulator of neuronal precursor activity and cognitive and motor behaviors (50); CFB, a complement factor altered in neurologic diseases such as multiple sclerosis (51) and HOXB3, a critical choreographer of neural development (52).The biological interpretation of these changes was also studied using gene set enrichment analysis (GSEA).We identified gene sets related to signaling pathways known to be regulated by T3 and relevant for cerebral cortex development, including MAPK, cAMP, Wnt, mTOR, TNF, TGFB, Hippo, and NFK (Figure 5K).
Next, we took advantage of our RNA-seq data to assess T3-regulated genes (53,54) proposed to have a role in cerebral cortex development (46) and noted significant expression changes between Control and MCT8-deficient COs (Figures 5L-O).We focused our attention on genes that are important for (a) normal development of the cerebral cortex, (b) neural cell migration, (c) astrocytes and myelination, and (d) neurotransmitters receptors, transcription factors, potassium channels, and extracellular matrix proteins (Figures 5L-O).
With respect to category (d), the genes included GABRA5 (neurotransmitter γ-amino butyric acid receptor subunit) and CHRNA5 (acetylcholine nicotinic receptor subunit).The transcription factors TOX3 and KLF6 (for neuron survival (61)).MYCN, EMX1, and ZHX2 (for progenitor cell division or maintenance (62)).The potassium channels, KCNC1, KCNK9, and KCNK1 (influencing the biology of neurons and astrocytes) and the genes ADAMTS2, GPC3, GPC6, BMP1, CRIM1, NAV2, L1CAM, and TNC (extracellular matrix components important for an array of developmental processes including neuronal migration and axon outgrowth ( 46)).In summary, among the genes studies in the four categories, 14 downregulated genes in MCT8-COs are known to be transcriptionally upregulated by T3, and 15 upregulated genes in MCT8deficient COs are known to be transcriptionally repressed by T3 (Supplemental table 7).These findings strongly support our previous results showing that the T3 transport and action during

Discussion
The discovery that mutations in MCT8 are associated with the AHDS revealed that transport across the blood-brain-barrier and cell membranes is required for TH action in the brain (7,63).
However, critical questions remained unresolved.Here, we answer some of these questions and provide direct experimental evidence that reducing T3 transport into human neural cells leads to impaired T3 signaling and underlies the pathophysiology of the neurologic manifestations of AHDS.Our experiments with TH-analogs demonstrated that DITPA and TRIAC could trigger improved responses (induction/repression of T3-responsive genes) in MCT8-deficient human neural cells, highlighting the promising potential for the early utilization of TH analogs in the treatment of patients with AHDS.
Having access to the human brain of MCT8-deficient patients would be the ultimate resource for the scientists who study the pathophysiology of this syndrome, but the invasive nature of the methods available limits such samples.Here, we resolved this hurdle by establishing a hiPSCsderived model of COs that re-creates an MCT8-deficient environment mimicking the early fetal human brain development.To our knowledge, this is the first generation and characterization of MCT8-deficient COs derived from hiPSCs obtained from AHDS patients (7).Some of the critical functional features noted are: i) EBs exhibited smooth and optically translucent edges demonstrating neural induction; ii) COs developed neuroepithelial bud expansion and formed cerebral-like structures of similar size; and iii) COs had multiple large and continuous small cortical units composed of a neural rosette arranged in a laminar fashion constituting neuroepithelium-like structures similar to a ventricle.In addition, Control and MCT8-deficient COs exhibited similar mRNA levels of forebrain markers and proliferative and pluripotency markers (Figure 1).The presence of these features in MCT8-deficient COs indicates that a nonfunctional MCT8 does not compromise the potential of neural cells to initiate neurodevelopment.However, as detailed herein, subsequent proliferation and differentiation of these precursors are altered in MCT8-deficient COs.
For our studies, we utilized isogenic control COs (isoWT) and control COs obtained from hiPSCs of the unaffected father of patient Mut2, with ~50% genetic similarity, in order to minimize the genetic background variance.Despite the difference in the genetic background between the two control lines, they showed similar outcomes in most of our experiments, indicating that the differences observed in MCT8-deficient COs are indeed due to the lack of functional MCT8.Our study is not without limitations as COs exhibit high heterogeneity, which may lead to low reproducibility.In our studies, this has led to variability in gene expression among replicates.Also, more physiological culture conditions are needed since the maturation of COs requires supraphysiological doses of T3 (60 nM) for long periods of time, which may partly overcome the lack of MCT8 by using alternative transporters Our findings support the hypothesis that an MCT8-mediated TH action is critical during early gestation, playing a role in human neurogenesis (43).This is evidenced by the smaller rosettes and thinner cortical units observed in MCT8-COs.Alterations in these morpho-functional patterns have been associated with impaired proliferation of neural progenitor cells and even microcephaly in different cellular and animal models (64)(65)(66)(67)(68).In agreement, we observed that the plane of divisions of the neural precursor cells was different in Control compared to MCT8deficient COs, being predominantly horizontal in Control COs.This finding is consistent with a study describing similar alterations in the neural progenitors during the early development of the optic tectum of chickens with a reduced expression of MCT8 (18).The presence of these alterations in MCT8-deficient COs highlights the need for MCT8-dependent TH uptake in neural precursor cells and clarifies the importance of local TH action to establish the layered structure of the human forebrain during early embryonic development.That these alterations may affect neural differentiation is further evidenced by the consequential reduced expression of the neural differentiation markers JAG1 and HES5 and of the neuronal markers TUBB3 and NEUN in MCT8-deficient COs and by a study analyzing brain and cerebellum sections from an MCT8deficient fetus that identified abnormalities in the density of neurons (8).Similar evidence was found in MCT8 knockin mice (P235L) that exhibit fewer neurons in the layers I-IV of the somatosensory cortex (69).
The present studies indicate that MCT8 is responsible for the bulk of the T3 transport across the membranes of the human neural cells residing in COs.This is illustrated by the reduced D3mediated T3-I 125 to T2-I 125 conversion in MCT8-deficient COs.The peak of T2-I 125 indicates the uptake of T3-I 125 into the neural cells, its metabolism, and the release of T2-I 125 to the medium.
In agreement, studies in MCT8-deficient neural cells show an altered uptake and efflux of TH (13).The low level of D3 metabolism observed in MCT8-deficient COs may be attributed to some residual MCT8 function or to additional transporters (70,71).The latter is less likely to be relevant, given (i) that SC blunted the D3 metabolism in the control line, resulting in low (but not fully suppressed) D3 activity, (ii) that Mut2 (missense mutation) exhibited more D3 activity than Mut1 (nonsense mutation; truncated protein) and (iii) that MCT8-deficient COs exhibited reduced MCT10, and LAT2 and similar LAT1 mRNA levels, suggesting an inability of the TH transport system to compensate for the absence of MCT8-dependent transport.This is consistent with observations in other MCT8-deficient iPSC-derived brain microvascular endothelial-like cells carrying a different mutation (72), although a decrease in mRNA levels of alternative transporters was not reported in iPSCs-derived MCT8-deficient neural cells (13).
Conversely, in Mct8 KO mice, the expression of alternative TH transporters increases (73), likely contributing to the absence of an obvious neurological phenotype in these animals.
We have found that D2 is active in the early stages of COs maturation when a high percentage of neural precursor cells expressing MCT8 is expected (Figure 3A, B; (16,74,75)).The D2generated T3 seems to be of great value for these cells, as illustrated by the fact that we could not find D2-generated T3-I 125 in the medium-as would be the case in mature astrocytes known to release the D2-generated T3 to the medium to act in neurons in a paracrine fashion (76,77)).
Instead, we found that the elevated expression of the cytoplasmatic protein µ-crystallin (CRYM; reduced in MCT8-COs; Figure 5J) likely functions to retain the D2-generated T3 in the cytoplasm of the neural precursor cells.The resulting build-up of T3 can trigger developmental programs in these cells.The relevance of this observation cannot be underestimated, as these neural precursor cells are the source of most human cortical neurons (78).Further research into the role of MCT8 and D2 in the biology of these cells is in progress.
These studies not only show that MCT8 is present in postmitotic neurons (VIMENTIN+ and TUJ1+ cells;) residing in the COs, but also show that T3 signaling is altered in MCT8-deficient COs, consistent with the observed reduction in TH transport.The lack of response to T3 of some of the genes studied in MCT8-deficient COs is significant.Conversely, in control COs, T3 resulted in the expected changes in up-and down-regulated genes.Some of our results differ from a study on MCT8-dependent neural cells that showed similar dose-dependent T3-induced gene expression (13).A possible explanation for this discrepancy is that our T3 treatment lasted 24h versus a ~14 days chronic exposure to T3 in the latter.A limitation in both studies is the use of relatively high T3 concentrations (nM range), which do not recapitulate the lower in vivo conditions of free TH in cells (pM range) (79).The high doses of T3 may explain the unexpected increase in the mRNA levels of the gene HAIRLESS (and, to a lesser extent, the gene KLF9) in the Mut1 COs.
Regulation of gene expression is the foundation of our understanding of how TH acts in the developing brain.Most of what we know is inferred from studies in hypothyroid animals (80), mouse primary cultures (53,54,81), and by expressing a mutated version of the T3 receptor in specific mouse cells (82,83).In our study, we have looked at ~65 T3-regulated genes that regulate critical processes of cerebral cortex development, and we found them to be altered in MCT8-deficient COs, making the case that TH signaling could be severely altered during the neurodevelopment of patients with MCT8-deficient syndrome.It is important to note that many of these genes are involved in critical functions, and the alterations of only a few of them can result in severe consequences-the control of CAMK4 expression is a good example.CAMK4 is known to be a transcriptional target of T3, and it is abundant in cortical neurons, having a crucial role in brain development and function (55,84).Among CAMK4 actions are the regulation of neuron differentiation and apoptosis and the regulation of dendritic growth and synaptic activity.
We found that CAMK4 was among the top downregulated genes in MCT8-deficient COs.
Conversely, we also found some genes whose expression patterns were unexpected (i.e., induction/repression by T3 and the same responses in MCT8-deficient COs).This is the case of genes such as MYCN and SLIT2, which were upregulated by T3 in primary cultures and were also found to be upregulated in MCT8-deficient COs.These scenarios represent evidence that, in most cases, it is not easy to describe T3 actions linearly; in contrast, T3 action during brain development contributes to a network of interconnected pathways.Altogether, these new findings in the transcriptome of Control and MCT8-deficient COs suggest that in the maturation of the human brain, the changes in the transcriptome caused by T3 signaling are indeed significant and depend on MCT8.
The present studies also advance our understanding of how the TH-analogs DITPA and TRIAC work in MCT8-deficient neural cells.The responses of the genes KLF9, CIRBP, and COL6A1 to these TH-analogs are significant.It was difficult to predict which TH-analog would be more effective since DITPA and TRIAC have no preference for any TRs.We found that both THanalogs elicited similar responses in both MCT8-deficient COs.In addition, the present study demonstrates that DITPA and TRIAC can enter human neural cells via an MCT8-independent mechanism.This is illustrated by the fact that the Mut2 COs and isoWT responded similarly to the same dose of the analogs.The physiological implications of these findings are considerable, as they validate our species-specific model for the early development of the brain in an MCT8deficient environment while showcasing the potential effectiveness of these TH-analogs.
Considering that (i) postnatal treatment with these analogs did not ameliorate the neurological symptoms, and that (ii) when treating a mother carrying an MCT8-deficient fetus from 18 weeks of gestation until birth at 35 weeks (85), it improved the neuromotor and neurocognitive function, our study supports the idea that early prenatal treatment with TH analogs that can be available to fetus when given to the mother (86) may further rescue the phenotype.
In conclusion, here we provided definitive evidence that MCT8 is critical for early neurogenesis, mediating the bulk of the T3 uptake into developing human neural cells.Such perturbations and altered mechanisms likely play a key role in the pathogenesis of AHDS.Our studies validate DITPA and TRIAC as treatments that can elicit thyromimetic responses in human MCT8deficient neural cells.Because of the inter-species differences in the expression of TH transporters in the brain, our studies using human MCT8-deficient COs carry significant physiological and clinical implications for how MCT8 loss-of-function mutations relate to the neurological manifestations seen in patients while providing the groundwork to develop new treatments.
Generation of human brain organoids.The methods to generate COs are highly reproducible (30,91).On day 0, 80%-confluent hiPSC were dissociated using the Gentle Cell Dissociation Reagent, and ~9000 cells/well were seeded in a 96-well ultra-low-attachment plate (Corning) in Embryoid bodies (Ebs) formation medium containing 10 µM Y-27632 (Tochris).On day 5, the medium was removed, and Ebs were incubated with a neural induction medium.On day 7, Ebs were kept on the 96-well plate, and the medium was supplemented with 2% Matrigel.On day 10, Ebs were transferred into ultra-low-attachment 6-well plates kept in a maturation medium on an orbital shaker (75 rpm), with media changes every other day.From day 20 onwards, the maturation medium was switched to COs maturation medium, containing: BrainPhys and the supplements SM1, N2A, NEAA (Gibco), Glutamax (Gibco), Insulin (Sigma), BME (EMD-Millipore) and 2 ng/ml BDNF (Tochris).Every three days, half of the medium was replaced with a fresh one.For experiments with TH-analogs, ~22 days old COs were incubated in a maturation medium supplemented with 1% stripped serum (92) and with either 10nM L-T3, 10nM TRIAC, or 3.5µM DITPA (doses were adjusted based on their potency on cultured cells).
Our D20 COs contained neural and glial precursor cells as well as dispersed neurons, establishing the structural and cellular framework for further maturation.To obtain COs populated by the three main cellular lineages of the brain, i.e., neurons, astrocytes, and oligodendrocytes, we followed established protocols (35,93) and treated COs from day 48 to 50 with platelet-derived growth factor AA and insulin-like growth factor 1 (10 ng/ml; Tochris) and from days 50 to 65 with T3 (60nM; Sigma); Figure 5A).Treatment periods mirror the initial specification of oligodendrocyte precursor cells and mature oligodendrocytes (the myelingenerating cells) in the fetal human brain, which occurs at gestational weeks 10 and 14, respectively (35,94).

Quantitative real-time PCR (qRT-PCR).
Total RNA was extracted from 4 COs per group and treatment.mRNAs were treated with Dnase and measured by quantitative RT-PCR as previously described (95).Briefly, total RNA was isolated using a Qiagen RNAeasy mini kit (Qiagen catalog #74104), according to the manufacturer's instructions.The cDNA was prepared using a cDNA synthesis kit (Roche).Data were analyzed using the 2 −ΔΔCT method and displayed relative to an arbitrary value.The expression of the indicated genes was determined using specific primers (Supplementary Table 1).The expression of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control.
RNA sequencing and analysis.Samples of total RNA was sent to the genomic facility at the University of Chicago for library preparation and sequence.Libraries were pair-end sequenced with NovaSeqS4 (Illumina).Base calls and demultiplexing were performed with Illumina's bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read.The FASTQ files were aligned to gencode hg38 transcriptome with STAR (v.2.7.8a) using the Partek Flow platform (Partek Inc., St. Louis, MO, USA).Aligned reads were quantified to the annotation model (Partek E/M) and normalized to counts per million.

Figure 4 .
Figure 4. TRIAC and DITPA can trigger TH signaling in MCT8-deficient COs.A-D.Changes in the mRNA levels of the indicated genes after 24h of the indicated treatments.The genes HAIRLESS and KLF9 are upregulated by T3, while the genes CIRBP and COL6A are downregulated (99, 100).values are mean ± SD of n = 5-6 RNA samples, each of them consisting of 4 pooled COs from either WT or MCT8-deficient COs; 1-way ANOVA and Tukey test were used for multiple comparisons; *P <0.05, **P < 0.01, ***P < 0.001; ns: non-significant.