Tsh Induces Agrp1 Neuron Proliferation in Oatp1c1-Deficient Zebrafish

Abstract

Thyroid hormones (THs), thyroxine (T4), and triiodothyronine (T3), regulate growth, metabolism, and neurodevelopment. THs secretion is controlled by the pituitary thyroid-stimulating hormone (TSH) and the hypothalamic-pituitary-thyroid (HPT) axis. The organic anion-transporting polypeptide 1C1 (OATP1C1/SLCO1C1) and the monocarboxylate transporter 8 (MCT8/SLC16A2) actively transport THs, which bind to their nuclear receptors and induce gene expression. A mutation in <i>OATP1C1</i> is associated with brain hypometabolism, gradual neurodegeneration, and impaired cognitive and motor functioning in adolescent patients. To understand the role of Oatp1c1 and the mechanisms of the disease, we profiled the transcriptome of <i>oatp1c1</i> mutant (<i>oatp1c1</i> ^-/-) and <i>mct8</i> ^-/- <i>xoatp1c1</i> ^-/- adult male and female zebrafish brains. Among dozens of differentially expressed genes, agouti-related neuropeptide 1 (<i>agrp1</i>) expression increased in <i>oatp1c1</i> ^-/- adult brains. Imaging in the hypothalamus revealed enhanced proliferation of Agrp1 neurons in <i>oatp1c1</i> ^-/- larvae and adults, and increased food consumption in <i>oatp1c1</i> ^-/- larvae. Similarly, feeding and the number of Agrp1 neurons increased in thyroid gland-ablated zebrafish. Pharmacological treatments showed that the T3 analog TRIAC (3,3',5-tri-iodothyroacetic acid), but not T4, normalized the number of Agrp1 neurons in <i>oatp1c1</i> ^-/- zebrafish. Since the HPT axis is hyperactive in the <i>oatp1c1</i> ^-/- brain, we used the CRISPR-Cas9 system to knockdown <i>tsh</i> in <i>oatp1c1</i> ^-/- larvae, and inducibly enhanced the HPT axis in wild-type larvae. These manipulations showed that Tsh promotes proliferation of Agrp1 neurons and increases food consumption in zebrafish. The results revealed upregulation of both the HPT axis-Agrp1 circuitry and feeding in a zebrafish model for OATP1C1 deficiency.<b>SIGNIFICANCE STATEMENT</b> Mutation in the thyroid hormone (TH) transporter <i>OATP1C1</i> is associated with cognitive and motor functioning disturbances in humans. Here, we used an <i>oatp1c1</i> ^-/- zebrafish to understand the role of organic anion-transporting polypeptide 1C1 (Oatp1c1), and the characteristics of OATP1C1 deficiency. Transcriptome profiling identified upregulation of <i>agrp1</i> expression in the <i>oatp1c1</i> ^-/- brain. The <i>oatp1c1</i> ^-/- larvae showed increased thyroid-stimulating hormone (<i>tsh</i>) levels, proliferation of Agrp1 neurons and food consumption. Genetic manipulations of the hypothalamic-pituitary-thyroid (HPT) axis showed that Tsh increases the number of Agrp1 neurons and food consumption. The T3 analog TRIAC (3,3',5-tri-iodothyroacetic acid) normalizes the number of Agrp1 neurons and may have potential for the treatment of Oatp1c1 deficiency. The findings demonstrate a functional interaction between the thyroid and feeding systems in the brain of zebrafish and suggest a neuroendocrinological mechanism for OATP1C1 deficiency.