Pathogenic variants in the GNAO1 gene, encoding the alpha subunit of an inhibitory heterotrimeric guanine nucleotide-binding protein (Go) highly expressed in the mammalian brain, have been linked to encephalopathy characterized by different combinations of neurological symptoms, including developmental delay, hypotonia, epilepsy and hyperkinetic movement disorder with life-threatening paroxysmal exacerbations. Currently, there are only symptomatic treatments, and little is known about the pathophysiology of GNAO1-related disorders. Here, we report the characterization of a new in vitro model system based on patient-derived induced pluripotent stem cells (hiPSCs) carrying the recurrent p.G203R amino acid substitution in Gαo, and a CRISPR-Cas9-genetically corrected isogenic control line. RNA-Seq analysis highlighted aberrant cell fate commitment in neuronal progenitor cells carrying the p.G203R pathogenic variant. Upon differentiation into cortical neurons, patients’ cells showed reduced expression of early neural genes and increased expression of astrocyte markers, as well as premature and defective differentiation processes leading to aberrant formation of neuronal rosettes. Of note, comparable defects in gene expression and in the morphology of neural rosettes were observed in hiPSCs from an unrelated individual harboring the same GNAO1 variant. Functional characterization showed lower basal intracellular free calcium concentration ([Ca²⁺]_i), reduced frequency of spontaneous activity, and a smaller response to several neurotransmitters in 40- and 50-days differentiated p.G203R neurons compared to control cells. These findings suggest that the GNAO1 pathogenic variant causes a neurodevelopmental phenotype characterized by aberrant differentiation of both neuronal and glial populations leading to a significant alteration of neuronal communication and signal transduction.

Organoids are three-dimensional (3D) cell cultures that replicate some of the key features of morphology, spatial architecture, and functions of a particular organ. Organoids can be generated from...

GNAO1 is an alpha subunit of the G-protein complex involved in signal transduction in neurons. The G203R mutation in the GNAO1 gene arises recurrently de novo and causes epileptic encephalopathy and movement disorder. GNAO1 has two main isoforms, GNAO1-A and GNAO1-B, but their functional or expression differences are poorly understood. Molecular functions of GNAO1 are mainly studied in neurons, yet glial cells also express GNAO1 and participate in the pathogenesis of epilepsy. Here, we used human-induced pluripotent stem cell-based models to investigate the localization and expression of GNAO1 isoforms in astrocytes. We showed that in astrocytes, almost 100% of GNAO1 transcripts encoded GNAO1-B with very low GNAO1-A expression. We showed that there were no differences in localization between GNAO1-A and GNAO1-B, both in WT and G203R states. We also showed that GNAO1 localized in astrocytic retraction fibers and migrasomes, structures not previously described in this cell type. We showed that GNAO1-positive retraction fibers of neighboring cells provided cell-to-cell contacts and also provided calcium waves during astrocytic excitation. Overexpression of both GNAO1-A and GNAO1-B tends to lower calcium activity in astrocytes, with GNAO1-A providing the most severe impairment of activity. Our results demonstrate that astrocytes, in addition to neurons, should be used as a model for studying GNAO1-related disorders and that GNAO1 mutations should be evaluated in the context of both the GNAO1-A and GNAO1-B isoforms.

BACKGROUND: The UBE2A protein belongs to the E2 family of ubiquitin-binding enzymes involved in the ubiquitination of substrate proteins. UBE2A mutations lead to congenital X-linked mental retardation syndrome-type Nascimento. How UBE2A participates in the central nervous system development is still unknown. AIM: To establish a cell model based on induced pluripotent stem cells (iPSCs) to study the molecular and cellular functions of UBE2A in neurogenesis. METHODS: Using genomic CRISPR-Cas9 editing and lentiviral transduction, a cell model based on iPSCs from two healthy donors was designed. This cell model includes isogenic iPSCs with knockout and inducible hyperexpression of UBE2A. In addition, iPSCs were obtained by reprogramming peripheral blood mononuclear cells of a patient diagnosed with X-linked mental retardation of Nascimento type, which has a deletion spanning the whole UBE2A locus. RESULTS: The obtained iPSCs demonstrate an ESC-like morphology. They express pluripotent cell markers OCT4, SOX2, SSEA-4, and TRA-1-81 and have normal karyotypes. iPSCs with UBE2A knockout or hyperexpression had significantly increased nuclei size compared with the isogenic control. CONCLUSION: The developed iPSC-based cell model can be used for fundamental studies of the functions of UBE2A in neurogenesis.