Ricerc@Sapienza

Fragile X syndrome (FXS) is an inherited form of human mental retardation caused by epigenetic silencing in the FMR1 gene and the consequent loss of the fragile X mental retardation protein (FMRP) is directly linked with alterations in dendritic spine morphology, synaptogenesis and connectivity in the developing brain leading to neurological, cognitive and behavioral defects.
Our study aims to create a 3D model based on human induced pluripotent stem cells (hiPSCs) with the purpose of deciphering the neurobiological phenotypes associated with FXS. In order to study the electrophysiological properties of FXS iPSC-derived neurons in-vitro, we firstly optimized a 2D differentiation protocol for obtaining a mixed cortical neuron culture. Although preliminary patch-clamp experiments showed no differences, in terms of Na+ and K+ currents and ability to evoke action potentials, between control and FXS neural cultures, calcium imaging analysis showed that the FXS lines appear to display a reduced frequency of spontaneous calcium events, with significantly reduced synchronicity at day 68, thus suggesting an alteration in the neuronal network development.
Moreover, in order to better characterize the alteration due to the absence of FMRP and reduce the genetic variability between iPSC lines, we generated an FMR1 knockout iPSC line, isogenic to the control one, by using a CRISPR gene-editing approach. These two populations will be then used for generating up an iPSC-derived 3D cell model based on the whole-brain organoids culture method.
Currently, we report the efficient production of brain organoids from three control hiPSC lines and we aim to further study how the absence of FMRP can morphologically and functionally invalidate the formation of cortical plates during early neurodevelopment events.