OR14-02 The Thyroid Hormone Analogs DITPA And TRIAC Triggered T3-signalling In Human MCT8-deficient Brain Organoids

Abstract Disclosure: F. Salas Lucia: None. A.M. Dumitrescu: None. S. Refetoff: None. Patients with mutations in the X chromosome-linked thyroid hormone (TH) cell transporter MCT8 gene have brain hypothyroidism, resulting in severe neuro-psychomotor retardation for which treatment remains very challenging. Potential new treatments using the TH-analogues 3,5-diiodothyropropionic acid (DITPA) and 3,3’,5-triiodothyroacetic acid (TRIAC) have been explored in Mct8-deficient mice. However, given species differences in brain TH transporters, in vitro models using human cells are needed. Here, we generated human brain organoids (BOs) from 4 lines of human induced pluripotent stem cells (hiPSCs). Two lines were derived from MCT8-deficient patients. The other two lines were used as controls: one isogenic line in which the MCT8 mutation has been corrected using CRISPR/Cas9, and a line derived from the unaffected father. Control BOs showed multiple large and continuous neuroepithelia surrounding a ventricle and expressed forebrain markers (FOXG1, and LHX2) at 10-fold higher levels than the hiPSCs. After 20 days in culture, BOs expressed TH transporters; the most abundant being MCT8, trailed by LAT2, LAT1, and MCT10. In contrast, the MCT8-deficient BOs exhibit a 3-fold decrease in the relative expression of LAT2, compared to controls. All BOs showed a similar expression of the deiodinase type 3 (DIO3), and the TH nuclear receptor beta (THRB) genes; the DIO2 and THRA expression were undetectable. Additionally, all four BOs expressed the neuronal and glial markers NEUN and GFAP (5.1±2.0, and 7.3±3.8-fold expression, respectively). Immunohistochemistry using fluorescent antibodies confirmed the presence of postmitotic neurons (TUJ1+) expressing MCT8 in the BOs. Next, we measured the D3 activity. BOs were incubated with T3I125, which resulted in a prominent peak of T2I125 measured using an ultra-high-performance liquid chromatography linked to a gamma counter. The peak of T2I125 indicates the uptake of T3I125 into the neural cells residing in the BOs, its metabolism, and the release of T2I125 in the medium. MCT8-deficient BOs exhibited a D3 activity of 5.3±3.1 pmol/mg/h, a 70% less when compared to the 18.4±7.7 pmol/mg/h of controls. When control BOs were incubated with the MCT8 inhibitor Silychristin 2µM, they exhibited 4.4±3.0 pmol/mg/h D3 activity, like MCT8-deficient BOs. Next, BOs were incubated for 24h with either 10nM L-T3, 10nM TRIAC, or 3.5μM DITPA and T3-responsive genes were studied. Treatment with L-T3 upregulated by 2-fold the gene KLF9 in all four BOs, suggesting that the T3-signalling in the MCT8-deficient BOs might not be compromised. The genes HAIRLESS and DIO3 were unresponsive. In parallel, incubation with DITPA and TRIAC also upregulated KLF9 by 1.5-fold, representing a proof of concept for these thyromimetic molecules to act in human neural cells. This human cell model may be used as a platform to screen therapeutic drugs for MCT8 deficiency. Presentation: Friday, June 16, 2023