Citterio_2025_Int.J.Mol.Sci_26_

Thyroid hormones (thyroxine, T(4), and triiodothyronine, T(3)) are indispensable for sustaining vertebrate life, and their deficiency gives rise to a wide range of symptoms characteristic of hypothyroidism, affecting 5-10% of the world's population. The precursor for thyroid hormone synthesis is thyroglobulin (Tg), a large iodoglycoprotein consisting of upstream regions I-II-III (responsible for synthesis of most T(4)) and the C-terminal CholinEsterase-Like (ChEL) domain (responsible for synthesis of most T(3), which can also be generated extrathyroidally by T(4) deiodination). Using CRISPR/Cas9-mediated mutagenesis, we engineered a knock-in of secretory ChEL into the endogenous TG locus. Secretory ChEL acquires Golgi-type glycans and is properly delivered to the thyroid follicle lumen, where T(3) is first formed. Homozygous knock-in mice are capable of thyroidal T(3) synthesis but largely incompetent for T(4) synthesis such that T(4)-to-T(3) conversion contributes little. Instead, T(3) production is regulated thyroidally by thyrotropin (TSH). Compared to cog/cog mice with conventional hypothyroidism (low serum T(4) and T(3)), the body size of ChEL-knock-in mice is larger; although, these animals with profound T(4) deficiency did exhibit a marked elevation of serum TSH and a large goiter, despite normal circulating T(3) levels. ChEL knock-in mice exhibited a normal expression of hepatic markers of thyroid hormone action but impaired locomotor activities and increased anxiety-like behavior, highlighting tissue-specific differences in T(3) versus T(4) action, reflecting key considerations in patients receiving thyroid hormone replacement therapy.