Endocrine Abstracts

Background: Disrupted thyroid hormone (TH) homeostasis has devastating effects on human neurodevelopment. THs are critical signaling molecules in neurodevelopment, acting on differentiation of neural cells, migration, synaptogenesis and myelination, with deiodinases governing intracellular TH concentrations in a spatiotemporal manner. It is remarkable that fetal neural cells, while being key target cells of TH, exhibit strong activity of the TH inactivating enzyme DIO3. Currently, the molecular mechanisms underlying TH action in brain are mainly derived from animal models. We utilized human induced pluripotent stem cell (hiPSC) technology to investigate the role of DIO3 in a human model for early brain development.

Methods: We generated a complete DIO3 knock-out (KO) using the CRISPR/Cas9 technology in hiPSCs. hiPSC were differentiated to neurons by NGN2 overexpression as a model for fetal human neurons. hiPSCs-derived neurons contained all the key players in TH cellular signaling. In parallel, we inactivated DIO3 in neural cells using iopanoic acid (IOP), a small molecule that blocks DIO3 activity. Cells were cultured with different T3 concentrations (0-3-10 nM). We used immunocytochemistry, gene expression and DIO3 and metabolism assays as readouts.

Results: CRISPR/Cas9-generated DIO3 KO neurons presented a complete absence of DIO3 activity. Upon T3 addition, there was an enhanced response in gene expression of T3-dependent genes such as KLF9 in DIO3 KO vs wild-type NGN2 neurons. Both outcomes were validated in IOP-treated neural cultures. We also examined the differentiation potential of neural progenitor cells to neural networks in absence of DIO3 by IOP treatment. Our preliminary immunocytochemistry data showed an increase in cells containing neuronal nuclear protein (NeuN), a biomarker for neurons, when the activity of DIO3 is diminished.

Conclusion: Our results suggest that impaired DIO3 activity may lead to excessive TH action in neural cells and, thereby, compromising normal brain development. We hypothesize that a high DIO3 activity is required in early brain development to maintain stemness and prevent premature neural differentiation. Our model represents a versatile tool to investigate cellular TH regulation and action not only for neural development but in other stem cell models.