FMR1 loss in a human stem cell model reveals early changes to intrinsic membrane excitability.

Fragile X mental retardation 1 (FMR1) encodes the RNA binding protein FMRP. Loss of FMRP drives Fragile X syndrome (FXS), the leading inherited cause of intellectual disability and a leading monogenic cause of autism. While cortical hyperexcitability is a hallmark of FXS, the reported phenotypes and underlying mechanisms, including alterations in synaptic transmission and ion channel properties, are heterogeneous and at times contradictory. Here, we report the generation of new isogenic FMR1y/+ and FMR1y/- human pluripotent stem cell (hPSC) lines using CRISPR-Cas9 to facilitate the study of how complete FMRP loss, independent of genetic background, drives molecular and cellular alterations relevant for FXS. After differentiating these stem cell tools into excitatory neurons, we systematically assessed the impact of FMRP loss on intrinsic membrane and synaptic properties over time. Using whole-cell patch clamp analyses, we found that FMR1y/- neurons overall showed an increased intrinsic membrane excitability compared to age-matched FMR1y/+ controls, with no discernable alternations in synaptic transmission. Surprisingly, longitudinal analyses of cell intrinsic defects revealed that a majority of significant changes emerged early following in vitro differentiation and some were not stable over time. Collectively, this study provides a new isogenic hPSC model which can be further leveraged by the scientific community to investigate basic mechanisms of FMR1 gene function relevant for FXS. Moreover, our results suggest that precocious changes in the intrinsic membrane properties during early developmental could be a critical cellular pathology ultimately contributing to cortical hyperexcitability in FXS.

Effects of Manipulating Fibroblast Growth Factor Expression on Sindbis Virus Replication In Vitro and in Aedes aegypti Mosquitoes.

Fibroblast growth factors (FGFs) are conserved among vertebrate and invertebrate animals and function in cell proliferation, cell differentiation, tissue repair, and embryonic development. A viral fibroblast growth factor (vFGF) homolog encoded by baculoviruses, a group of insect viruses, is involved in escape of baculoviruses from the insect midgut by stimulating basal lamina remodeling. This led us to investigate whether cellular FGF is involved in the escape of an arbovirus from mosquito midgut. In this study, the effects of manipulating FGF expression on Sindbis virus (SINV) replication and escape from the midgut of the mosquito vector Aedes aegypti were examined. RNAi-mediated silencing of either Ae. aegypti FGF (AeFGF) or FGF receptor (AeFGFR) expression reduced SINV replication following oral infection of Ae. aegypti mosquitoes. However, overexpression of baculovirus vFGF using recombinant SINV constructs had no effect on replication of these viruses in cultured mosquito or vertebrate cells, or in orally infected Ae. aegypti mosquitoes. We conclude that reducing FGF signaling decreases the ability of SINV to replicate in mosquitoes, but that overexpression of vFGF has no effect, possibly because endogenous FGF levels are already sufficient for optimal virus replication. These results support the hypothesis that FGF signaling, possibly by inducing remodeling of midgut basal lamina, is involved in arbovirus midgut escape following virus acquisition from a blood meal.