Deciphering the mechanism of impaired neurogenesis in fragile X syndrome

Introduction: Fragile X Syndrome (FXS) is the main genetic cause for mental retardation. FMRP is expressed in FXS embryos and inactivated by the end of the first trimester. Neural differentiation of FXS human embryonic stem cells (hESC) mimics the neurodevelopment of FXS fetuses and thus serves as a good model to explore the mechanisms underlining the development of FXS.

Aim: To elucidate the molecular events underlying the aberrant function of FXS neurons.

Methods: FX-hESCs sub-clones with and without the FX mutation sharing the same genetic background were generated, verified using PCR based repeat number assay, and subjected to in vitro neuronal differentiation. Lines were compared by immunofluorescence for neuronal markers and FMR1 expression and RNA sequencing was performed. Magnetic sorting for neurons enrichment was executed using the cell surface markers CD24 and CD184.

Results: Within 13 days of differentiation Tuj1⁺ neurons were already obtained. FX-hESCs demonstrated delayed differentiation as compared to WT and showed lower FMR1 expression at their pluripotent stage that was completely silenced in neurons. Neurons generated from full-mutation sub-clones silenced FMRP, compared to the normal controls that expressed FMRP. Magnetic sorting was able to enrich the neurons population.

Conclusions: Neurons were generated from hESCs already within 13 days. Neurons derived from FX full-mutation hESCs mimicked the gradual downregulation of FMR1 as seen in human FXS fetuses. These unique set of clones are currently undergoing RNA sequencing, results that will open the door for further molecular analysis of the effects of FMR1 dysregulation on the pathophysiology underlying FXS.