SAT-387 - Local thyroid hormone inactivation by deiodinase type 3 affects neuronal identity acquisition and maturation

Thyroid hormone (T3) promotes neurogenesis, and this study aims to determine T3 effects on neuronal identity acquisition and maturation. We developed a live/fixed correlative imaging method to directly map progenitor division and the generation of newborn neurons in human iPSC-derived cultures. Through live analysis, we identified hundreds of dividing progenitor cells (SOX2+) and intermediate progenitors (EOMES+) during the first nine days in culture (D9). Using a neuronal-specific probe (NeuO), we observed peak neurogenesis on D10, when newborn neurons began expressing the type 3 deiodinase (DIO3) as early as 10 hours after birth. However, newborn progenitors did not express DIO3. Preliminary studies of progenitors with a non-functional MCT8 T3 transporter showed that newborn MCT8-deficient neurons are more likely to undergo apoptosis than controls. To understand how DIO3 expression changes during neuronal maturation, we used RT-qPCR and observed a steady increase in DIO3 mRNA levels in neuronal cultures at D10, D20, and D40. Consistently, DIO3 enzymatic activity progressively increased, reaching in D40 neurons extremely high values (200 pmol/h/mg; ~100 times higher than in mouse primary neurons). Notably, these D40 neurons exhibited spontaneous electrical activity and calcium signals. Since DIO3’s function is to reduce T3 levels, we cultured differentiating progenitors in a medium containing B26, a homemade supplement lacking T3. Remarkably, progenitors differentiated into neurons and showed a steady increase in DIO3 mRNA levels at D10, D20, and D40. Notably, neurons differentiated in B26 + T3 (60nM) exhibited similar levels of DIO3 expression. Notwithstanding, D40 neurons on B26 + T3 exhibited reduced mRNA levels of GluN2A and the postsynaptic protein PSD95, suggesting a maturation delay. These findings indicate that DIO3 expression is a critical component of the developmental program of newborn neurons, likely associated with the expression of genes within the epigenetically regulated DLK1-DIO3 imprinted domain, including DLK1, MEG3, MEG8, and RTL1, all of which increased their expression to a peak in D40 neurons. Targeted bisulfite sequencing of this domain identified 25 hypermethylated CpG sites in D40 neurons compared with progenitors, 3 of them in MEG3 and MEG8, 1 in the DIO3 promoter, and the remaining 21 in the RTL1 gene body. Considering DIO3’s function in reducing T3 availability in newborn and maturing neurons, a corollary of these findings is that T3’s role in neurogenesis may be mediated by its epigenetically regulated action on neural progenitors rather than as a critical gatekeeper of neuronal identity acquisition and maturation.

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