ИСТИНА

The G203R mutation in the GNAO1 is a rare genetic condition that can lead to severe neurological disorders. Using iPSC from a patient with this mutation, the study investigated the pathogenesis of GNAO1-encephalopathy. iPSC were differentiated into neural lineages and stimulated to form 3D-neural rosettes. The G203R/+ neural progenitor cells produced small clusters of cells with abnormal structures. Time-lapse microscopy detected no issues with cell migration during neuronal rosette formation. xCELLigence analysis found a two-fold increase in cell index for the G203R culture compared to the control. Calcein staining revealed a significant increase in area occupancy for G203R cells, with aberrant structures forming. Immunocytochemical analysis using the neuronal markers βIII-tubulin and MAP2 revealed the presence of positively stained cells in aberrant structures. However, the quantity of these cells was significantly different from that of neural rosettes in the control culture. Pax6+ cell analysis revealed a significant deviation, with predominantly cytoplasmic localization in the G203R culture, unlike the nucleus-specific localization in the control culture. Neuronal cells were examined for intracellular calcium signaling in response to neurotransmitters, including GABA, acetylcholine, clonidine, glutamine, and fentanyl. The mutant culture showed a radical increase in the calcium concentration upon adding acetylcholine, a bidirectional effect in response to clonidine, and no significant response to glutamate, fentanyl, and GABA. In conclusion, GNAO1-encephalopathy may be caused by a violation of brain morphogenesis and Gαo signaling dysfunction during life contributing to the progression of this rare neurodevelopmental disorder.