Previous work in animal models of fragile X syndrome (FXS) has provided invaluable insight into the normal molecular, cellular, and physiological functions of fragile X mental retardation protein (FMRP); however, an effective treatment remains elusive. Although these failures could be attributed to several factors, it is now apparent that it is imperative that FXS-associated phenotypes, the efficacy of drugs, and rescue strategies characterized in animal models of FXS be validated and/or new phenotypes characterized in human FXS patient- derived, disease-relevant cell types. A critical limitation is lack of an available human FXS patient-derived neural model to investigate the role of FMRP-mediated regulation of protein synthesis and signaling. We have recently developed multiple human iPSC-derived 2D neural and 3D cortical organoid models to investigate the role of FMRP-mediated regulation of protein synthesis and signaling during brain development. The objectives of Project 1 are to use these FXS patient iPSC-derived 2D monolayers as well as 3D cortical and hippocampal organoids to address questions delineated in three specific aims.
Aim 1 is to characterize protein synthesis dysregulation and associated molecular, cellular and neurophysiological phenotypes in specific cell types across neural development in human FXS iPSC neural models. Our preliminary data indicate that FXS patient cells have increased protein synthesis rates, increased proliferation and altered migration, resulting in delayed acquisition of cell fate and neuronal differentiation. These early neurodevelopmental defects are anticipated to have consequences on neuronal development and function.
Aim 2 is to identify FMRP targets and translationally dysregulated mRNAs during brain development in multiple human FXS iPSC neural models. Using CLIP-seq we have identified FMRP target mRNAs in both human cortical organoids and mouse embryonic cortex at similar developmental stages. Our comparative analyses have revealed three groups of FMRP mRNA targets, human only, mouse only and shared ones. We have also recently used ribosome profiling to identify translationally dysregulated mRNAs, some of which are FMRP targets, in whole cortex in the adult mouse brain. Thus, ribosome profiling will be applied to characterize the translatomes of FXS patients and controls using both isogenic i3Neurons and i3Neurons from multiple patients, as well as from isogenic 3D cortical organoids. For comparison between FXS models, we also will conduct ribosome profiling of FXS mouse embryonic cortex.
In Aim 3, we will devise targeted strategies to rescue cellular and synaptic phenotypes in human FXS iPSC neural models. We will manipulate expression of dysregulated FMRP targets using lentivirus-based approaches to rescue FXS- associated cellular and synaptic phenotypes. The outcome of the experiments in this Project, coupled with synergy with the other projects, will uncover novel mechanisms and key drivers of FXS-associated phenotypes in cortical development using our newly generated human iPSC-derived 2D and 3D neural models.
