Transcriptomic Analysis of Human Fragile X Syndrome Neurons Reveals Neurite Outgrowth Modulation by the TGFß/BMP Pathway.

Fragile X Syndrome (FXS) is the main genetic reason for intellectual disability and is caused by the silencing of fragile X mental retardation protein (FMRP), an RNA-binding protein regulating the translation of many neuronal mRNAs. Neural differentiation of FX 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. Isogenic hESC clones with and without the FX mutation that share the same genetic background were in vitro differentiated into neurons, and their transcriptome was analyzed by RNA sequencing. FX neurons inactivating FMR1 expression presented delayed neuronal development and maturation, concomitant with dysregulation of the TGFß/BMP signaling pathway, and genes related to the extracellular matrix. Migration assay showed decreased neurite outgrowth in FX neurons that was rescued by inhibition of the TGFß/BMP signaling pathway. Our results provide new insights into the molecular pathway by which loss of FMRP affects neuronal network development. In FX neurons, the lack of FMRP dysregulates members of the BMP signaling pathway associated with ECM organization which, in a yet unknown mechanism, reduces the guidance of axonal growth cones, probably leading to the aberrant neuronal network function seen in FXS.

Impaired Functional Connectivity Underlies Fragile X Syndrome.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by a developmentally regulated silencing of the FMR1 gene, but its effect on human neuronal network development and function is not fully understood. Here, we isolated isogenic human embryonic stem cell (hESC) subclones-one with a full FX mutation and one that is free of the mutation (control) but shares the same genetic background-differentiated them into induced neurons (iNs) by forced expression of NEUROG-1, and compared the functional properties of the derived neuronal networks. High-throughput image analysis demonstrates that FX-iNs have significantly smaller cell bodies and reduced arborizations than the control. Both FX- and control-neurons can discharge repetitive action potentials, and FX neuronal networks are also able to generate spontaneous excitatory synaptic currents with slight differences from the control, demonstrating that iNs generate more mature neuronal networks than the previously used protocols. MEA analysis demonstrated that FX networks are hyperexcitable with significantly higher spontaneous burst-firing activity compared to the control. Most importantly, cross-correlation analysis enabled quantification of network connectivity to demonstrate that the FX neuronal networks are significantly less synchronous than the control, which can explain the origin of the development of intellectual dysfunction associated with FXS.

Functional Deficiencies in Fragile X Neurons Derived from Human Embryonic Stem Cells.

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is a neurodevelopmental disorder caused by silencing of the FMR1 gene, which in FXS becomes inactivated during human embryonic development. We have shown recently that this process is recapitulated by in vitro neural differentiation of FX human embryonic stem cells (FX-hESCs), derived from FXS blastocysts. In the present study, we analyzed morphological and functional properties of neurons generated from FX-hESCs. Human FX neurons can fire single action potentials (APs) to depolarizing current commands, but are unable to discharge trains of APs. Their APs are of a reduced amplitudes and longer durations than controls. These are reflected in reduced inward Na(+) and outward K(+) currents. In addition, human FX neurons contain fewer synaptic vesicles and lack spontaneous synaptic activity. Notably, synaptic activity in these neurons can be restored by coculturing them with normal rat hippocampal neurons, demonstrating a critical role for synaptic mechanisms in FXS pathology. This is the first extensive functional analysis of human FX neurons derived in vitro from hESCs that provides a convenient tool for studying molecular mechanisms underlying the impaired neuronal functions in FXS. SIGNIFICANCE STATEMENT: Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by silencing of the FMR1 gene. In this study, we describe for the first time the properties of neurons developed from human embryonic stem cells (hESCs) that carry the FMR1 mutation and are grown in culture for extended periods. These neurons are retarded compared with controls in several morphological and functional properties. In vitro neural differentiation of FX hESCs can thus serve as a most relevant system for the analysis of molecular mechanisms underlying the impaired neuronal functions in FXS.

Heterozygous APC germline mutations impart predisposition to colorectal cancer.

Familial adenomatous polyposis (FAP) is an inherited syndrome caused by a heterozygous adenomatous polyposis coli (APC) germline mutation, associated with a profound lifetime risk for colorectal cancer. While it is well accepted that tumorigenic transformation is initiated following acquisition of a second mutation and loss of function of the APC gene, the role of heterozygous APC mutation in this process is yet to be discovered. This work aimed to explore whether a heterozygous APC mutation induces molecular defects underlying tumorigenic transformation and how different APC germline mutations predict disease severity. Three FAP-human embryonic stem cell lines (FAP1/2/3-hESC lines) carrying germline mutations at different locations of the APC gene, and two control hESC lines free of the APC mutation, were differentiated into colon organoids and analyzed by immunohistochemistry and RNA sequencing. In addition, data regarding the genotype and clinical phenotype of the embryo donor parents were collected from medical records. FAP-hESCs carrying a complete loss-of-function of a single APC allele (FAP3) generated complex and molecularly mature colon organoids, which were similar to controls. In contrast, FAP-hESCs carrying APC truncation mutations (FAP1 and FAP2) generated only few cyst-like structures and cell aggregates of various shape, occasionally with luminal parts, which aligned with their failure to upregulate critical differentiation genes early in the process, as shown by RNA sequencing. Abnormal disease phenotype was shown also in non-pathological colon of FAP patients by the randomly distribution of proliferating cells throughout the crypts, compared to their focused localization in the lower part of the crypt in healthy/non-FAP patients. Genotype/phenotype analysis revealed correlations between the colon organoid maturation potential and FAP severity in the carrier parents. In conclusion, this study suggest that a single truncated APC allele is sufficient to initiate early molecular tumorigenic activity. In addition, the results hint that patient-specific hESC-derived colon organoids can probably predict disease severity among FAP patients.

Modeling FXS: Human Pluripotent Stem Cells and In Vitro Neural Differentiation.

In fragile X syndrome (FXS) embryos FMRP is widely expressed during early stages of embryogenesis however it is inactivated by the end of the first trimester. In the same manner, human embryonic stem cell (hESC) lines from FXS blastocysts, bearing the full CGG expansion mutation, express FMRP in their pluripotent stage and in neurons derived following in vitro differentiation, FMR1 is completely silenced. Therefore, in vitro neural differentiation of FX-hESC lines serves as a uniquely valuable model system to study the developmental mechanisms underlying FXS, together with the proper differentiation protocol to mimic the neurodevelopmental process occurs in vivo.