FMR1 loss results in early changes to intrinsic membrane excitability in human cellular models

Fragile X mental retardation 1 (FMR1) encodes the RNA binding protein FMRP. Loss of FMRP drives Fragile X syndrome (FXS), the leading inherited cause of intellectual disability and a leading monogenic cause of autism. Cortical hyperexcitability is a hallmark of FXS, however, the underlying mechanisms reported, including alterations in synaptic transmission and ion channel expression and properties, are heterogeneous and at times contradictory. Here, we generated isogenic FMR1y/+ and FMR1y/- human pluripotent stem cell (hPSC) lines using CRISPR-Cas9, differentiated these stem cell tools into excitatory cortical neurons and systematically assessed the impact of FMRP loss on intrinsic membrane and synaptic properties over the course of in vitro differentiation. Using whole-cell patch clamp analyses at five separate time-points, we observed significant changes in multiple metrics following FMRP loss, including decreased membrane resistance, increased capacitance, decreased action potential half-width and higher maximum frequency, consistent with FMR1y/- neurons overall showing an increased intrinsic membrane excitability compared with age-matched FMR1y/+ controls. Surprisingly, a majority of these changes emerged early during in vitro differentiation and some were not stable over time. Although we detected significant differences in intrinsic properties, no discernable alterations were observed in synaptic transmission. Collectively, this study provides a new isogenic hPSC model to study the mechanisms of FMR1 gene function, identifies electrophysiological impacts of FMRP loss on human excitatory cortical neurons over time in vitro, and underscores that early developmental changes to intrinsic membrane properties may be a critical cellular pathology contributing to cortical hyperexcitability in FXS.