Investigating neural development in Fragile X Syndrome using human iPSC models

Abstract

Fragile X Syndrome (FXS) is a neurodevelopmental disorder characterised by a variety of symptoms from intellectual disability to anxiety, sensory hypersensitivity, epilepsy and autistic behaviour. It is caused by a loss of expression of the FMRP protein, resulting from a CGG repeat expansion in the FMR1 gene 5’UTR. There are currently no effective treatments available for FXS. Previous studies in mouse and human FMR1 knockout (KO) models of FXS suggest that loss of FMRP expression affects the function of neural progenitor cells (NPCs) in the developing brain. The aim of this study was to investigate how loss of FMRP expression affects the ability of FXS NPCs to proliferate, migrate, and give rise to neurones, as well as to characterise the proteomic landscape of FXS patient iPSC-derived NPCs. This was done through generation of isogenic controls for three FXS patient-derived iPSC lines through CRISPR/Cas9-mediated removal of the pathogenic CGG repeat, leading to reactivation of FMRP expression in these cells. These isogenic control cells and their FXS parental lines were then differentiated into functional neural progenitors (NPCs).

Proteomic analysis of the FXS and control NPCs showed that loss of FMRP led to alterations in signalling pathways important for neuronal development, and cellular processes such as mRNA transcription and editing, autophagy and mitochondrial function. When these NPCs were differentiated into neurones, there were alterations in PAX6 expression during early differentiation, and a reduction in the presynaptic puncta formed along their neurites. Some previously established FXS-associated phenotypes could not be replicated in the NPCs of the presentstudy, which may have been due to inter-platedown and inter-conversion variation that was observed in these datasets. Examination of human FMRP-deficient astrocytes showed that these cells may also be affected by the loss of FMRP expression, both in their GFAP protein expression and at the transcriptomic level. Altogether, these data show for the first time that loss of FMRP leads to alterations in FXS patient iPSC-derived NPCs and FMRP-deficient astrocytes as compared to their respective isogenic controls. They will provide a starting point for further studies elucidating the pathomechanisms of and potential treatments for FXS.