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

Abstract

Patients with mutations in the thyroid hormone (TH) cell transporter monocarboxylate transporter 8 (MCT8) gene develop severe neuropsychomotor retardation known as Allan-Herndon-Dudley syndrome (AHDS). It is assumed that this is caused by a reduction in TH signaling in the developing brain during both intrauterine and postnatal developmental stages, and treatment remains understandably challenging. Given species differences in brain TH transporters and the limitations of studies in mice, we generated cerebral organoids (COs) using human induced pluripotent stem cells (iPSCs) from MCT8-deficient patients. MCT8-deficient COs exhibited (i) altered early neurodevelopment, resulting in smaller neural rosettes with thinner cortical units, (ii) impaired triiodothyronine (T3) transport in developing neural cells, as assessed through deiodinase-3-mediated T3 catabolism, (iii) reduced expression of genes involved in cerebral cortex development, and (iv) 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 COs, constituting proof of concept that lack of T3 transport underlies the pathophysiology of AHDS and demonstrating the clinical potential for TH analogs to be used in treating patients with AHDS. MCT8-deficient COs represent a species-specific relevant preclinical model that can be utilized to screen drugs with potential benefits as personalized therapeutics for patients with AHDS.

Keywords: Endocrinology; Neurodevelopment; Neuroscience; Thyroid disease; Transport.

Figure 1. Generation and characterization of control and MCT8-deficient COs.

(A) Schematic of COs’ generation and timing. COs’ generation started with the culture of EBs, followed by neural induction, neuroepithelial bud expansion, and maturation of the COs. Arrows point to expanded neuroepithelia as evidenced by the EB surface budding. (B) Mut1, Mut2, and isoWT COs exhibit neuroepithelial expansion. (C and D) Control and MCT8-deficient COs formed cerebral like structures (C) of similar size (D). (E and F) Relative gene expression (normalized to GAPDH) as determined by quantitative PCR from D20 COs of CNS markers (FOXG1, NKX2.1, LMX1B) (E) and of pluripotency (SOX2) and ectoderm (OCT4) markers (F); n = 5–6 RNA samples, each of them consisting of 4 pooled COs from either WT or MCT8-deficient COs. Two-tailed Student’s test for comparing human iPSCs versus human COs, and 1-way ANOVA and Tukey test were used for multiple comparisons (growth and SOX2 expression between COs); ***P < 0.001.

Figure 2. MCT8-deficient COs exhibit impaired early neurodevelopment and reduced expression of neuronal markers.

(A) Confocal images of cryostat 10 μm–thick slice of a WT D20 CO. The arrow points to dense vertical columns of SOX2/Ki67⁺ cells, including a fluid-filled luminal compartment with an apical layer of progenitors (double asterisk and arrowheads in the inset). Scale bar 75 μm; inset in A: scale bar 35 μm. (B) Whole-mount immunolabeling of D20 control and MCT8-deficient COs. Scale bar 500 μm. (C) Representative circular rosette-like substructures (nuclear staining DAPI in blue). ** indicates areas of undifferentiated tissue; scale bar 150 μm. (D and E) Quantitation of the diameter of the cortical rosettes and number of layers formed by SOX2⁺ cells. Scale bar 150 μm; inset in D: scale bar 50 μm. Note how both parameters are reduced in MCT8-deficient COs when compared with controls. Values are mean ± SD of 4 COs per line (5–6 rosettes per CO). (F) Staining for phospho-histone H3 (PH3; red) and phospho-Vimentin (green) to mark neural precursor cells in mitosis, which primarily divide at the apical surface. Arrows in F and in the insets mark apical surface horizontal (0–30 degrees) and vertical (60–90 degrees) divisions. Scale bar 10 μm; insets in F: scale bar 5 μm. (G) Quantitation of neural precursors’ division orientation from 4 COs per line. (H) Whole-mount immunolabeling of D20 COs, Tuj1 staining in green, nuclear staining DAPI (blue) showing evenly distributed postmitotic neurons. Scale bar 100 μm; and insets in H: scale bar 150 μm. (I) Quantitation of the mRNA levels of the indicated genes in D20 control and MCT8-deficient COs. Note how the expression of genes involved in neurogenesis and neuronal differentiation is reduced in MCT8-deficient when compared with control COs. 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.

Figure 3. MCT8 mediates T3 transport in human neural cells.

(A and B) Whole-mount immunolabeling of D20 WT COs, vimentin staining in green, MCT8 in red, colocalization indicated as yellow, nuclear staining DAPI as blue (A). (B) TUJ1 staining in green, MCT8 in red, nuclear staining DAPI in blue. Scale bar in A: 100 μm, in B: 10 μm. MCT8 staining was present in the outer cell membrane of neuronal elements (arrows). (C) Quantitation of the mRNA levels of the indicated TH transporters in D20 control and MCT8-deficient COs. (D) Same as C but for the genes THRA and THRB. (E) Same as C but for the genes DIO3 and DIO2. (E) Same as C but for the gene DIO3. (F) Representative chromatograms of the medium after WT, Mut1, and Mut2 COs were incubated with T4-I¹²⁵ for 3 hours. (G) Quantitation of the D3 deiodination in the indicated conditions; n = 5–13 D3 assays. (H) Same as in C but for the gene DIO2. (I) Relative DIO2 mRNA levels in WT COs during their first 20 days in culture. (J) T4-I¹²⁵ deiodination in D15 and D20 CO sonicates. (K) Relative CRYM mRNA levels in WT COs at D15 and D20. Expression values are mean ± SD of n = 3–6 RNA samples, each of them consisting of 4 pooled COs from either WT or MCT8-deficient COs; WT+SC: WT incubated with T4-I¹²⁵ in the presence of 2 μM of Silychristin (SC). Two-tailed Student’s test for comparing D2 deiodination and relative mRNA expression between D15 and D20, and 1-way ANOVA and Tukey test were used for multiple comparisons; *P < 0.05, **P < 0.01, ***P < 0.001.

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 24 hours 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.

Figure 5. MCT8-deficient COs present an altered transcriptome.

(A) Schematic of the protocol for COs’ maturation using platelet-derived growth factor AA and insulin-like growth factor 1. (B–E) Whole-mount immunolabeling of D65 WT COs containing differentiated NEUROD1⁺ neurons (B), GFAP⁺ neural precursor cells and GFAP-AQP4⁺ astrocytes (C and D), and OLIG2⁺ oligodendrocyte precursor cells and O4⁺ premyelinating oligodendrocytes (E); in blue is the nuclear staining with DAPI. B: scale bar 75 μm; C: scale bar 75 μm; inset in C: scale bar 25 μm; D: scale bar 40 μm; E: scale bar 75 μm; inset in E: scale bar 40 μm. Arrows in D indicate AQP4 staining in the membrane of GFAP⁺ cells. The arrow in the inset in E points to the cellular body of a representative oligodendrocyte; arrowheads point to its cellular processes. (F and G) Volcano plots showing the distribution of DEGs in WT vs. Mut1 (F) and in WT vs. Mut2 (G); each point represents the average of 3 (WT) or 2 (MCT8-deficient) samples consisting of 4 pooled COs for each transcript. (H) Venn comparison of WT vs. Mut1, WT vs. Mut2, and Mut1 vs. Mut2 DEGs. A common cluster (cluster E) between WT vs. Mut1 and WT vs. Mut2 containing 949 DEGs was identified. (I) Venn comparison of the 949 DEGs in cluster E shows similar transcriptomic changes in both MCT8-deficient COs. (J) Heatmap depicting the top 63 DEGs from cluster E. (K) KEGG pathway analysis of the top 63 DEGs in J. (L–O) Heatmaps of T3-regulated genes involved in cerebral cortex development (L), neural cell migration (M), astrocytes and myelination (N), and neurotransmitter receptors, transcription factors, potassium channels, and extracellular matrix protein (O). DEG thresholds (FDR ≤ 0.25 and fold-change ± 4) were identified by gene set–specific analysis in the Partek Flow platform.