Ricerc@Sapienza

Several models have been developed in order to provide significant understanding of FXS and various targets for potential pharmaceutical treatments have been identified, many of which have been shown to be efficient in preclinical studies. However, all attempts to turn these findings into a therapy for patients did not give the desired results; furthermore, animal models cannot recapitulate the mechanism of FMR1 silencing nor the potential regulatory mechanisms that may be involved during neural development. So the generation of animal models that completely mimic the developmental progress of FXS at the molecular and cellular level has been challenging.
In 2006 Yamanaka and his colleagues revolutionized disease modeling by successfully reprogramming murine fibroblast to a pluripotent cell type, so called induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006). One year later they succeeded to reproduce the protocol for human fibroblasts (Takahashi et al., 2007) and in the past ten years technologies to generate iPSCs have vastly improved. Next to this, there have been many technical advances enabling the differentiation from pluripotent cells towards relatively pure populations of specific neuronal subtypes including excitatory neurons, specific subtypes of inhibitory neurons, astrocytes, oligodendrocytes and microglia. So growing evidence demonstrates how aspects of human development and disease can be accurately modelled in vitro using hiPSCs (Passier et al., 2016). With the purpose of deciphering the molecular mechanisms and the neurobiological phenotypes associated with FXS, the aim of this project is the creation of a robust in vitro model based on hiPSCs which can be used to study FXS in a time frame that is relevant to the disease, understanding its mechanisms and allow for therapeutic testing in cells carrying the genetic background of individual patients. Two types of neurons could be used as in vitro models of FXS: cortical neurons and hippocampal neurons. We are currently setting up conditions for neuronal differentiation starting from human iPSC lines with the Fragile X syndrome mutation and one line with the FMR1 premutation (range from 55 to 200 repeats). Previous work by the Bhattacharyya lab demonstrated that FXS-iPSCs can be differentiated into FOXG1+ forebrain neurons (Doers et al., 2014) and the mutation does not impair the differentiation capacity of these cells. It has been demonstrated that FXS-iPSC-derived forebrain neurons exhibited neurite outgrowth defects, in particular FXS forebrain neurons extend significantly fewer processes that are significantly shorter relative to controls (Doers et al., 2014). So, once obtained a robust method of differentiation, we want to study the electrophysiological properties and axon growth dynamics of FXS-iPSCs derived neurons and the possible rescue function obtained by GSK3b inhibitors. No data are currently available on the electrophysiological properties of FXS-iPSCs derived neurons and in particular we want to analyze voltage gated channels and firing properties of FXS-iPSCs derived neurons and synaptic activity on neuronal networks in vitro, using patchclamp and ion imaging recordings.
Therefore, the creation of a robust in vitro model based on hiPSCs can efficiently be used to study the molecular mechanisms of FXS, to recapitulate the disease progression and it could serve for therapeutic testing. Furthermore the use of patient derived iPSCs provide an unlimited supply of material for molecular, proteomic and electrophysiological studies as well as enabling the observation of normally inaccessible neurodevelopmental processes.