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Experimental Procedures
Author Contributions
Acknowledgments
Introduction Fragile X syndrome (FXS) affects approximately 1 in every 4,000 boys and 1 in 8,000 girls worldwide (Callan and Zarnescu, 2011; Penagarikano et al., 2007). It is now believed that FXS is the leading cause of inherited intellectual disability in males and one of the major monogenic causes for autism (Boyle and Kaufmann, 2010; Callan and Zarnescu, 2011; Penagarikano et al., 2007; Wang et al., 2012). The syndrome is caused primarily by an expansion of a CGG repeat at the 5′ untranslated region (UTR) of the fragile X mental retardation gene 1 (FMR1). This repeat expansion leads to CpG methylation, which spreads to the FMR1 promoter, modifications in chromatin conformation of the FMR1 gene, and silencing of the gene expression. Subsequently, the fragile X mental retardation protein (FMRP) is no longer produced (Coffee et al., 1999, 2002; Sutcliffe et al., 1992). FMRP is a highly conserved protein, expressed in mammals mainly in the Z-IETD-FMK and testes (Devys et al., 1993; Santoro et al., 2012; Verkerk et al., 1991). In the brain, FMRP is found primarily in neurons, where it plays an important role in synaptic plasticity (Devys et al., 1993). FMRP is an RNA-binding protein that acts as a translation regulator by either stalling polyribosomes or inhibiting translation initiation (Ashley et al., 1993; Feng et al., 1997; Khandjian et al., 2004; Napoli et al., 2008; Stefani et al., 2004). It may also regulate mRNA levels through the microRNA (miRNA) pathway, as work on both Drosophila and mammalian cells revealed association of FMRP with components of the RNA-induced silencing complex and several miRNAs (Caudy et al., 2002; Ishizuka et al., 2002; Jin et al., 2004; Plante et al., 2006). FMRP was also shown to associate with specific miRNAs, which together select and repress target mRNAs to regulate neuronal morphology (Edbauer et al., 2010). Several works have implicated a role for FMRP in neurogenesis, and although some of the results were contradicting, all of these studies have shown impairment in dendritic spine morphology, maturation or pruning, or abnormal gene expression during neural development that may persist to adulthood (Bhattacharyya et al., 2008; Castrén et al., 2005; Comery et al., 1997; Galvez et al., 2005; Irwin et al., 2001; Tessier and Broadie, 2008). Other studies have shown FMRP to be crucial for the regulation of timing and proliferation capacities of neural progenitor cells (NPCs), thus regulating the proper number of neurons (Callan et al., 2010; Egger et al., 2008; Luo et al., 2010). All of these data place FMRP as an important regulator of proper development and maturation of the neural network. Another key factor important for proper brain development is the repressor element 1 silencing transcription factor (REST) (Chen et al., 1998). REST is considered a master negative regulator of neurogenesis, regulating the pool size and timing of differentiation of different neural lineages (Chen et al., 1998; Covey et al., 2012; Satoh et al., 2013; Schoenherr and Anderson, 1995). REST is expressed in embryonic stem cells (ESCs), NPCs, and nonneuronal cells, where it suppresses neuron-specific genes, in contrast to differentiated neurons where it is silenced (Chen et al., 1998; Schoenherr and Anderson, 1995). REST both regulates and is regulated by brain specific miRNAs and has been implicated to be involved in pluripotency and neurodegenerative pathologies (González-Castañeda et al., 2013; Gopalakrishnan, 2009; Hermanson, 2008; Marullo et al., 2010; Ooi and Wood, 2007; Zuccato et al., 2003). We have previously generated both ESCs and induced pluripotent stem cells (iPSCs) derived from FXS patients (Bar-Nur et al., 2012; Eiges et al., 2007; Urbach et al., 2010). Although the functions of FMRP have been studied extensively, the underlying molecular mechanisms causing the severe neuronal phenotypes are still largely unknown. In this study, we aim to understand the molecular pathology underling FXS using FXS-derived iPSCs, NPCs, and neurons. Our study suggests a major role for REST in the molecular pathology of FXS neurons. A better understanding of the developmental processes dysregulated in FXS will help in the search for a treatment to alleviate or even correct some of the abnormal molecular phenotypes.