FMRP-Mediated Gene Regulation in Human Brain Development

ABSTRACT: Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and a leading genetic cause of autism. FXS is caused by the loss of functional fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein forming a messenger ribonucleoprotein complex with polyribosomes in the regulation of protein synthesis at synapses. Three-dimensional (3D) aggregate culture of human-induced pluripotent stem cells (iPSCs) has evolved from embryoid body cultures, quite faithfully following human organogenesis, and provides a new platform to investigate human brain development in a dish, otherwise inaccessible to experimentation. To determine whether the loss of FMRP could alter the development of human brain organoids, we have generated forebrain organoids from three FXS male patients and three healthy male controls. We observed reduced proliferation of neural progenitor cells and premature neural differentiation as well as perturbed cell cycle progression in FXS forebrain organoids. There is also a deficit in the production of GABAergic neurons as well as an altered balance between the number of excitatory and inhibitory neurons in FXS organoids. Interestingly these deficits were not observed with FXS mouse model. To compare the differential gene expression caused by the loss of FMRP between human and mouse, we then performed RNA-seq to identify the differentially expressed genes using both mouse embryonic brain cortex and human forebrain organoids at the comparable developmental stages. We detected very few genes differentially expressed in the absence of Fmrp in mouse. However, we identified 200 genes downregulated and 126 genes up-regulated in human FXS organoids, indicating human-specific impact caused by the loss of FMRP. Our bulk RNA-seq analyses revealed a more pervasive gene expression alteration in human organoids.  Thus, it is plausible to speculate that there could be important human-specific genes that might be related with FXS pathogenesis. To test this idea, we used human forebrain organoids and the mouse embryonic brains in parallel to perform enhanced crosslinking and FMRP immunoprecipitation followed by high-throughput sequencing and bioinformatics analysis (CLIP-seq). Our analyses identified more than 3,700 mRNA bound by FMRP in human organoids. Of the 3,700 mRNAs, ~1,600 overlapped with mouse mRNAs. Further, ~80% of the identified mouse mRNA species overlap with previously published results in mouse. To better understand the developmental, cellular and molecular changes and to gain insight beyond bulk gene expression analysis in forebrain organoids from FXS patients compared to control, we next performed single cell RNA-seq (scRNA-seq) to characterize distinct cell populations, specific cell types and cell states. We found that the population of specific neuron type were changed upon the loss of FMRP. Next, investigated if there were developmental differences in the transitioning from the early, primitive state to one of the more committed end states between control and FXS organoids, with many cells distributed along a “trajectory” between them. we found several bifurcation points of FXS cells from the general trajectory, and the cells of these subpopulations with altered neuronal states are strongly enriched for FXS. These results together suggest that the loss of FMRP could cause neurodevelopmental deficits specifically in human, and fragile X organoids could provide a unique platform to study the molecular pathogenesis of FXS and identify human-specific druggable targets for FXS and autism in general. 

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE146878 | GEO | 2021/06/19

REPOSITORIES: GEO