glycogen phosphorylase We did not observe an increased

We did not observe an increased number of ubiquitin IBs in neurons derived from an isogenic iPSC line with a classical premutation length. It is likely that the expression of mRNA with longer CGG repeat lengths in UFM neurons results in accelerated pathological development compared with premutation neurons. Furthermore, in contrast to a single large intra-nuclear IB observed in FXTAS mouse models or in postmortem brain samples of FXTAS patients, we found multiple IBs in both cytoplasm and nucleus. The iPSC-derived neurons represent an early developmental stage in comparison with an aging brain. Developmental progression of the number and size of the inclusions is reported in FXTAS mice (Wenzel et al., 2010). Therefore, the pattern of ubiquitin inclusions observed here may reflect an early stage of the IB formation at the onset of the disease. Additionally we detected that both premutation and UFM neurons show increased numbers of FMRP aggregate-like structures (Figure 6C). FMRP has been reported to have a tendency to aggregate and spontaneously misfold toward β-rich structures in vitro (Sjekloca et al., 2011). Therefore, aggregation of FMRP may be contributing to the FXTAS pathology. Overall, our data provide evidence for an increased accumulation of ubiquitin and FMRP inclusions in UFM iPSC-derived neurons, which may be signs of an accelerated FXTAS phenotype in the UFM lines compared with the classical premutation. In summary, our analyses reveal that UFM individuals have not lost the ability to silence FMR1, but the size of the CGG expansion triggering the silencing is higher than the one described in FXS patients (200 CGG). Furthermore, inter-individual variability in the CGG size requited for silencing is present not only in UFM but also in two FXS patients analyzed in this study. We propose a model in which the threshold size together with the proportion of the FMR1 glycogen phosphorylase below this threshold delineates UFM and FXS phenotypes. UFM do not silence FMR1 and are spare of FXS pathology; nevertheless, our data suggest that the expression of FMR1 gene with large CGG expansion may increase their risk of developing FXTAS.
Experimental Procedures
Author Contributions
Acknowledgments We thank Kathrin Wagner, Daniel Kaiser, and Andreas Katopodis from Novartis Institutes for Biomedical Research (NIBR) for purification of PBMCs and isolation of T cells. We thank Shola Richards, Ieuan Clay, Caroline Gubser Keller, Sarah Brasa, Remi Terranova, and Ivan Galimberti from NIBR for discussions on data analysis and interpretation. We thank Yi Yang from NIBR for Cas9-gRNA vector. We thank Stephen Helliwell for comments on the manuscript. Research in the E.T., P.C., and G.N. laboratories was funded by Telethon grant to E.T. (GGP15257A).
Introduction Cells in the human body can be broadly classified as two major types, germline and somatic cells. The fusion of fully differentiated germ cells (i.e., sperm and oocyte) produces a totipotent zygote. Since all cells harbor the same genetic code, differentiation depends on the epigenetic state of each cell; germ cells reprogram epigenetic information during their specification, development, and maturation to acquire toti- or pluripotency. Germ cells in mice are specified from proximal epiblast cells that lack expression of some subsets of pluripotency genes such as Nanog, Kruppel-like factor 2 (Klf2), and T cell leukemia/lymphoma 1 (Tcl1), which are regulated by the transcription factors B lymphocyte-induced maturation protein 1 (BLIMP1), PR domain-containing 14 (PRDM14), and transcription factor AP-2 gamma (TFAP2C) (Nakaki et al., 2013; Ohinata et al., 2005, 2009; Weber et al., 2010; Yabuta et al., 2006; Yamaji et al., 2008). Specified PGCs reactivate pluripotency-associated genes and can be used to derive embryonic germ cells (EGCs) via stimulation with three cytokines, basic fibroblast growth factor (bFGF), stem cell factor (SCF), and leukemia inhibitory factor (LIF) (Matsui et al., 1992). Migrating PGCs in the hindgut replace repressive DNA methylation and H3K9 methylation marks with a repressive H3K27 trimethylation mark in their genome during migration (Seki et al., 2005, 2007). DNA demethylation occurs as a two-step process: the first demethylation wave occurs in a genome-wide manner during migration and a second wave occurs in a locus-specific manner, for example at germline-specific genes and imprinted loci at the time when PGCs arrive at the gonads (Seisenberger et al., 2012). CpG methylation marks can be removed via replication-dependent and independent mechanisms (Wu and Zhang, 2010). The former is regulated by inhibition of DNA methyltransferase activity during de novo DNA synthesis, whereas the latter (also known as active demethylation) is triggered by oxidation of 5-methylcytosine catalyzed by ten-eleven translocation (TET) proteins, which is followed by base excision repair (BER) (Hackett et al., 2012). PGCs use both active and passive demethylation to erase genome-wide methylation marks (Hajkova et al., 2008; Kagiwada et al., 2013; Kawasaki et al., 2014; Ohno et al., 2013; Popp et al., 2010). Global hypomethylation is induced in the transition from metastable embryonic stem cells (ESCs) cultured with serum plus LIF to ground-state ESCs cultured with 2i plus LIF, which is similar to the first wave of DNA demethylation in migratory PGCs (Ficz et al., 2013).