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Background: Congenital hypothyroidism screening traditionally relies on detecting elevated thyroid-stimulating hormone levels, yet this approach may not detect a specific type of congenital hypothyroidism caused by iodotyrosine dehalogenase-1 (Dehal1) deficiency. The deficiency of this enzyme prevents the deiodination of mono-iodotyrosine (MIT) and di-iodotyrosine (DIT) in the process of iodine recycling. This underscores the potential use of iodotyrosine or its metabolites as non-invasive urinary biomarkers for early diagnosis of congenital hypothyroidism. However, the urinary metabolites of MIT/DIT have not yet been discovered. Thus, this study aimed to identify the urinary metabolites of iodotyrosine in experimental models., Method: Gas chromatography mass spectrometry was used to identify the urinary metabolites of iodotyrosine following intraperitoneal injection of MIT in rats. An isotope dilution mass spectrometric assay was developed for assessment of identified metabolites. Urine samples from Dehal1 knockout mice were used to confirm the results., Results: We identified novel iodotyrosine metabolites, 3-iodo-4-hydroxyphenylacetic acid (IHPA), and 3,5-diiodo-4-hydroxyphenylacetic acid (Di-IHPA) as the primary urinary metabolites of MIT and DIT respectively. The concentrations of urinary IHPA and Di-IHPA were significantly higher in Dehal1 knockout mice., Conclusion: Our findings suggest that IHPA is detected in larger quantities and may hold more clinical significance than previously identified biomarkers like MIT and DIT, making it a promising candidate for diagnosing congenital hypothyroidism or other conditions associated with iodine recycling inhibition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Many cellular processes are governed by protein-protein interactions that require tight spatial and temporal regulation. Accordingly, it is necessary to understand the dynamics of these interactions to fully comprehend and elucidate cellular processes and pathological disease states. To map de novo protein-protein interactions with time resolution at an organelle-wide scale, we developed a quantitative mass spectrometry method, time-resolved interactome profiling (TRIP). We apply TRIP to elucidate aberrant protein interaction dynamics that lead to the protein misfolding disease congenital hypothyroidism. We deconvolute altered temporal interactions of the thyroid hormone precursor thyroglobulin with pathways implicated in hypothyroidism pathophysiology, such as Hsp70-/90-assisted folding, disulfide/redox processing, and N-glycosylation. Functional siRNA screening identified VCP and TEX264 as key protein degradation components whose inhibition selectively rescues mutant prohormone secretion. Ultimately, our results provide novel insight into the temporal coordination of protein homeostasis, and our TRIP method should find broad applications in investigating protein-folding diseases and cellular processes., (© 2024. The Author(s).)

Thyroid hormones are crucial for brain development and their deficiency during fetal and postnatal periods can lead to mood and cognitive disorders. We aimed to examine the consequences of thyroid hormone deficiency on anxiety-related behaviors and protein expression of hippocampal glutamate transporters in congenital hypothyroid male offspring rats. Possible beneficial effects of treadmill exercise have also been examined. Congenital hypothyroidism was induced by adding propylthiouracil (PTU) to drinking water of pregnant Wistar rats from gestational day 6 until the end of the weaning period (postnatal day 28). Next, following 4 weeks of treadmill exercise (5 days per week), anxiety-related behaviors were examined using elevated plus maze (EPM) and light/dark box tests. Thereafter, protein expression of astrocytic (GLAST and GLT-1) and neuronal (EAAC1) glutamate transporters were measured in the hippocampus by immunoblotting. Hypothyroid rats showed decreased anxiety-like behavior, as measured by longer time spent in the open arms of the EPM and in the light area of the light/dark box, compared to control rats. Hypothyroid rats had significantly higher GLAST and GLT-1 and lower EAAC1 protein levels in the hippocampus than did the euthyroid rats. Following exercise, anxiety levels decreased in the euthyroid group while protein expression of EAAC1 increased and returned to normal levels in the hypothyroid group. Our findings indicate that thyroid hormone deficiency was associated with alterations in protein expression of glutamate transporters in the hippocampus. Up-regulation of hippocampal GLAST and GLT-1 could be at least one of the mechanisms associated with the anxiolytic effects of congenital hypothyroidism., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Methods: Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) ( p  < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.

Thyroid hormones (THs) regulate gene expression by binding to nuclear TH receptors (TRs) in the cell. THs are indispensable for brain development. However, we have little knowledge about how congenital hypothyroidism in neurons affects functions of the central nervous system in adulthood. Here, we report specific TH effects on functional development of the cerebellum by using transgenic mice overexpressing a dominant-negative TR (Mf-1) specifically in cerebellar Purkinje cells (PCs). Adult Mf-1 mice displayed impairments in motor coordination and motor learning. Surprisingly, long-term depression (LTD)-inductive stimulation caused long-term potentiation (LTP) at parallel fiber (PF)-PC synapses in adult Mf-1 mice, although there was no abnormality in morphology or basal properties of PF-PC synapses. The LTP phenotype was turned to LTD in Mf-1 mice when the inductive stimulation was applied in an extracellular high-Ca 2+ condition. Confocal calcium imaging revealed that dendritic Ca 2+ elevation evoked by LTD-inductive stimulation is significantly reduced in Mf-1 PCs but not by PC depolarization only. Single PC messenger RNA quantitative analysis showed reduced expression of SERCA2 and IP 3 receptor type 1 in Mf-1 PCs, which are essential for mGluR1-mediated internal calcium release from endoplasmic reticulum in cerebellar PCs. These abnormal changes were not observed in adult-onset PC-specific TH deficiency mice created by adeno-associated virus vectors. Thus, we propose the importance of TH action during neural development in establishing proper cerebellar function in adulthood, independent of its morphology. The present study gives insight into the cellular and molecular mechanisms underlying congenital hypothyroidism-induced dysfunctions of central nervous system and cerebellum.

Thyroid hormones are critical for the development of opsins involved in color vision. Hypothyroid mice show delayed M-opsin development and expanded distribution of S-opsin on the retina. However, the effects of maternal hypothyroidism on opsin development remain unknown. This study investigates the effects of congenital central hypothyroidism and maternal hypothyroidism on opsin development in thyrotropin-releasing hormone knockout (TRH-/-) mice. We examined the mRNA expression and protein distribution of S/M-opsin on postnatal days (P)12 and 17, as well as mRNA expression of type 2 and 3 iodothyronine deiodinase (DIO2 and DIO3, respectively) in the retina and type 1 iodothyronine deiodinase (DIO1) in the liver at P12 in TRH+/- mice born to TRH+/- or TRH-/- dams, and conducted S/M-opsin analysis in TRH+/+ or TRH-/- mice born to TRH+/- dams at P12, P17, and P30. M-opsin expression was lower in TRH+/- mice born to TRH-/- dams than in those born to TRH+/- dams, whereas S-opsin expression did not significantly differ between them. DIO1, DIO2, and DIO3 mRNA expression levels were not significantly different between the two groups; therefore, thyroid function in peripheral tissues in the pups was similar. S/M-opsin expression did not significantly differ between the TRH+/+ and TRH-/- mice born to TRH+/- dams on any postnatal day. These results demonstrate that maternal hypothyroidism causes M-opsin developmental delay during the early developmental stages of neonatal mice, and TRH-/- mice, a model of congenital central hypothyroidism, born to a euthyroid dam do not have delayed opsin development.

Background: The sodium/iodide symporter (NIS) mediates active iodide accumulation in the thyroid follicular cell. Autosomal recessive iodide transport defect (ITD)-causing loss-of-function NIS variants lead to dyshormonogenic congenital hypothyroidism due to deficient iodide accumulation for thyroid hormonogenesis. Here, we aimed to identify, and if so to functionally characterize, novel ITD-causing NIS pathogenic variants in a patient diagnosed with severe dyshormonogenic congenital hypothyroidism due to a defect in iodide accumulation in the thyroid follicular cell, as suggested by nondetectable radioiodide accumulation in a normally located thyroid gland, as well as in salivary glands. Methods: The proposita NIS-coding SLC5A5 gene was sequenced using Sanger sequencing. In silico analysis and functional in vitro characterization of the novel NIS variants were performed. Results: Sanger sequencing revealed novel compound heterozygous SLC5A5 gene variants (c.970-3C>A and c.1106A>T, p.D369V). In silico analysis suggested that c.970-3C>A disrupts the canonical splice acceptor site located in intron 7. Splicing minigene reporter assay revealed that c.970-3C>A causes exon 8 skipping during NIS pre-mRNA splicing leading to the NIS pathogenic variant p.Y324H fs *148. Moreover, in silico analysis indicated p.D369V as pathogenic. Functional in vitro studies demonstrated that p.D369V NIS does not mediate iodide accumulation, as p.D369V causes NIS to be retained in the endoplasmic reticulum. Mechanistically, we propose an intramolecular ionic interaction involving the β carboxyl group of D369 and the guanidinium group of R130, located in transmembrane segment 4. Of note, an Asp residue at position 369-which is highly conserved in SLC5A family members-is required for functional NIS expression at the plasma membrane. Conclusions: We uncovered a critical intramolecular interaction between R130 and D369 required for NIS maturation and plasma membrane expression. Moreover, we identified the first intronic variant causing aberrant NIS pre-mRNA splicing, thus expanding the mutational landscape in the SLC5A5 gene leading to dyshormonogenic congenital hypothyroidism.