Depletion of the Fragile X Mental Retardation Protein in Embryonic Stem Cells Alters the Kinetics of Neurogenesis

Abstract

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and a leading cause of autism. FXS is due to the silencing of the Fragile X Mental Retardation Protein (FMRP), an RNA binding protein mainly involved in translational control, dendritic spine morphology and synaptic plasticity. Despite extensive studies, there is currently no cure for FXS. With the purpose to decipher the initial molecular events leading to this pathology, we developed a stem-cell-based disease model by knocking-down the expression of Fmr1 in mouse embryonic stem cells (ESCs). Repressing FMRP in ESCs increased the expression of amyloid precursor protein (APP) and Ascl1. When inducing neuronal differentiation, βIII-tubulin, p27 , NeuN, and NeuroD1 were upregulated, leading to an accelerated neuronal differentiation that was partially compensated at later stages. Interestingly, we observed that neurogenesis is also accelerated in the embryonic brain of Fmr1-knockout mice, indicating that our cellular model recapitulates the molecular alterations present in vivo. Importantly, we rescued the main phenotype of the Fmr1 knockdown cell line, not only by reintroducing FMRP but also by pharmacologically targeting APP processing, showing the role of this protein in the pathophysiology of FXS during the earliest steps of neurogenesis. Our work allows to define an early therapeutic window but also to identify more effective molecules for treating this disorder. Stem Cells 2017;35:374-385.

Modeling Fragile X syndrome in neurogenesis: An unexpected phenotype and a novel tool for future therapies

“…15,16 Starting from these considerations and from the fact that an effective specific therapy for FXS is not available yet, a mouse embryonic stem cell line displaying a reduced expression of Fmr1 by stable transfection of a specific shRNA directed against Fmr1 (shFmr1 ES) has been generated. 17 These cells do not display morphological abnormalities and cell cycle variations, however altered expression of a subset of genes mainly involved in neuronal differentiation and maturation determines a subjacent molecular pathology. Indeed, stimulating the differentiation of shFmr1 ES cells into the neuronal lineage results in an accelerated generation of neural progenitors and neurons during the first steps of differentiation.…”

“…Interestingly, neurogenesis is also accelerated in the embryonic brains of Fmr1 KO mice, indicating that the shFmr1 ES cell model recapitulates the molecular and cellular alterations present in vivo. 17 This phenotype in shFmr1 ES cells is likely due to an elevated level of the Amyloid Precursor Protein (APP), whose mRNA is a known target of FMRP. 18 APP is processed by the BACE-1 enzyme, producing the b-amyloid (Ab) peptide that is known to accelerate neurogenesis by activating the expression of achaete-scute family bHLH transcription factor 1 (Ascll).…”

“…19,20 It is interesting to point out that the increased level of Ab peptide in Fmr1-depleted ES cells induces the expression of Ascll. 17 This latter factor has a pivotal role in neuronal differentiation 21 and its induction in shFmr1 ES cells represents a surprising event and the key point to explain the subsequent altered neuronal differentiation. 17 Consistently, the cell phenotype is rescued not only by re-expressing human FMR1, but also by reducing the processing of APP by the specific BACE-1 inhibitor LY2811376.…”

“…17 This latter factor has a pivotal role in neuronal differentiation 21 and its induction in shFmr1 ES cells represents a surprising event and the key point to explain the subsequent altered neuronal differentiation. 17 Consistently, the cell phenotype is rescued not only by re-expressing human FMR1, but also by reducing the processing of APP by the specific BACE-1 inhibitor LY2811376. The importance of the Ab peptide in the physiopathology of FXS, as well as in other forms of autism and ID, has been extensively studied.…”

“…Depletion of FMRP alters the normal kinetics of neuronal differentiation. 17 We can speculate that this event uncoordinates different brain maturation pathways and programs, leading to subtle architectural abnormalities of several brain regions and, ultimately, to intellectual deficit.…”