The clinical availability of tissue-specific biomarkers of thyroid hormone (TH) action constitutes a “holy grail” for the field. Scientists have investigated several TH-dependent markers, including the tissue content of triiodothyronine (T3)—the active form of TH. The study of animal models and humans indicates that the T3 content varies among different tissues, mostly due to the presence of low-affinity, high-capacity cytoplasmic T3 binding proteins. Nonetheless, given that T3 levels in the plasma and tissues are in equilibrium, T3 signaling is defined by the intracellular free T3 levels. The available techniques to assess tissue T3 are invasive and not clinically applicable. However, the tracer kinetic studies revealed that serum T3 levels can accurately predict tissue T3 content and T3 signaling in most tissues, except for the brain and pituitary gland. This is true not only for normal individuals but also for patients with hypo or hyperthyroidism–but not for patients with non-thyroidal illness syndrome. Given this direct relationship between serum and tissue T3 contents and T3 signaling in most tissues, clinicians managing patients with hypothyroidism could refocus attention on monitoring serum T3 levels. Future clinical trials should aim at correlating clinical outcomes with serum T3 levels.

Background: Normal brain development, mood, and cognitive functions depend on thyroid hormone (TH) action. However, little is known about how TH mediates its actions in the human brain. This is due to limited access to human brains deprived of TH during fetal and early postnatal life, as well as from adults with altered thyroid status. One way to partially bypass these limitations is by using magnetic resonance imaging and spectroscopy, two neuroimaging techniques that provide detailed, noninvasive information on human brain structure and function. Another way is using human-induced pluripotent stem cell (hiPSCs)-derived three-dimensional in vitro systems, known as brain organoids, which allow for the study of fundamental aspects of the early stages of human brain development. Summary: This narrative review focuses on neuroimaging and brain organoid studies. Neuroimaging of human brains performed in individuals with different thyroid conditions provides information on the volume, myelination, blood flow, neural activity, and connectivity of different areas. Such studies show that suboptimal thyroid status can impact human brain development and its normal function throughout life. This is true not only for patients with sporadic congenital hypothyroidism, during pregnancy or early after birth, but also for adult patients with hypo- or hyperthyroidism, patients carrying mutations that manifest as impaired sensitivity to TH, and even for normal individuals during aging. Studies using brain organoids generated from hiPSCs of healthy individuals or patients with thyroid genetic conditions provide insights into how TH can impact the early development of the human cerebral cortex. Conclusions: The developmental alterations in children born to mothers with different degrees of gestational hypothyroidism or who developed hypothyroidism early in life are remarkable, affecting multiple brain regions and pathways, including the cerebral cortex, hippocampus, cerebellum, interhemispheric and corticospinal tracts, and associative nuclei. The data connecting such changes to poor neurological outcomes in adult patients with hypothyroidism represent an objective link between thyroid-specific functional brain alterations and behavior. Growing brain organoids require TH, which is critical for human neurogenesis and oligodendrogenesis. These models have proven useful in screening drugs with potential therapeutic effects for patients with genetic thyroid diseases.

The development of the brain, as well as mood and cognitive functions, are affected by thyroid hormone (TH) signaling. Neurons are the critical cellular target for TH action, with T3 regulating the expression of important neuronal gene sets. However, the steps involved in T3 signaling remain poorly known given that neurons express high levels of type 3 deiodinase (D3), which inactivates both T4 and T3. To investigate this mechanism, we used a compartmentalized microfluid device and identified a novel neuronal pathway of T3 transport and action that involves axonal T3 uptake into clathrin-dependent, endosomal/non-degradative lysosomes (NDLs). NDLs-containing T3 are retrogradely transported via microtubules, delivering T3 to the cell nucleus, and doubling the expression of a T3-responsive reporter gene. The NDLs also contain the monocarboxylate transporter 8 (Mct8) and D3, which transport and inactivate T3, respectively. Notwithstanding, T3 gets away from degradation because D3’s active center is in the cytosol. Moreover, we used a unique mouse system to show that T3 implanted in specific brain areas can trigger selective signaling in distant locations, as far as the contralateral hemisphere. These findings provide a pathway for L-T3 to reach neurons and resolve the paradox of T3 signaling in the brain amid high D3 activity.

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.

The regulation of thyroid activity and thyroid hormone (TH) secretion is based on feedback mechanisms that involve the anterior pituitary thyrotropin (TSH) and medial basal hypothalamus (MBH) TSH-releasing hormone (TRH). Plasma T3 levels can be “sensed” directly by the anterior pituitary and MBH, plasma T4 levels require local conversion of T4 to T3, which is mediated by the type 2 deiodinase (D2). To study D2-mediated T4 to T3 conversion and T3 production in the anterior pituitary gland, we used mouse pituitary explants incubated with ¹²⁵I-T4 for 48 h to measure T3 production at different concentrations of free T4. The results were compared to cultures of D1 or D2-expressing cells, as well as freshly isolated mouse tissue. These studies revealed a unique regulation of the D2 pathway in the anterior pituitary gland, distinct from that observed in non-pituitary tissues. In the anterior pituitary, increasing T4 levels reduced D2 activity slightly but caused a direct increase in T3 production. However, the same changes in T4 levels decreased T3 production in human HSkM cells and murine C2C12 cells (both skeletal muscle) and mouse bone marrow tissue, which reached zero at 50pM free T4. In contrast, the increase in T4 levels caused the pig kidney LLC-PK1 cells and kidney fragments to proportionally increase T3 production. These findings have important implications for both physiology and clinical practice, as they clarify the mechanism by which fluctuations in plasma T4 levels are transduced in the anterior pituitary gland to mediate the TSH feedback mechanism.

PURPOSE: The development of the selective RET inhibitors selpercatinib and pralsetinib has revolutionized the treatment of metastatic progressive RET-mutant medullary thyroid carcinoma (MTC) and other RET-driven cancers, given their more favorable side-effect profile. The aim of this study is to investigate the mechanisms of selpercatinib-induced thyroid dysfunction in athyreotic patients with RET-mutant MTC and in patients with RET-mutant non–small-cell lung cancer (NSCLC) who had a functional thyroid. MATERIALS AND METHODS: Thyroid hormone levels were evaluated in an observational cohort of five athyreotic patients with MTC and 30 patients with NSCLC before and after initiation of selpercatinib. In vitro experiments to identify the mechanism of selpercatinib-induced thyroid dysfunction were conducted in cells expressing endogenous D1, D2, and D3 iodothyronine deiodinases. RESULTS: Upon initiating treatment with selpercatinib, athyreotic patients developed clinical hypothyroidism with approximately 60% lower T3 levels despite adequate levothyroxine supplementation, whereas in patients with NSCLC, who retain a normal thyroid, selpercatinib resulted in a more attenuated reduction in serum T3, which was dose-dependent. We conducted studies in cells endogenously expressing either D1, D2, or D3, the three iodothyronine deiodinases. Selpercatinib inhibited D2-mediated T3 production in MSTO-211 cells by 50%. A modest repression of D2 mRNA was present in human thyroid cancer TT cells that express RET, but not in the MSTO-211 cells that do not. No effect of the drug was observed on D1 (activating deiodinase) or D3 (inactivating deiodinase). Thus, a nontranscriptional effect of selpercatinib on D2 activity is the most plausible explanation for the low T3 levels. CONCLUSION: An off-target effect of selpercatinib on D2-mediated T3 production leads to clinical hypothyroidism, primarily in levothyroxine-treated athyreotic patients. Liothyronine supplementation was needed to achieve normal T3 levels and restore clinical euthyroidism.