The GNAO1-B Splice Variant Is the Predominant Isoform in Human Astrocytes and Localizes to Retraction Fibers and Migrasomes

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.