Dissecting singularity within complexity: cell-type-specific nascent proteome contribution in Fragile X Syndrome patient-derived brain organoids

Fragile X Syndrome (FXS) is a leading cause of inherited intellectual disability. It is caused by the abnormal accumulation of CGG repeats at the 5’ end of the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene, leading to gene silencing early in development, and the consequent loss of its protein product named FMRP. This is an RNA-binding protein crucial for negative regulation of mRNAs translation associated with a multitude of cell signaling behaviors, including synaptic plasticity and dendritic spine architecture. The timing of silencing as well as the cell types affected remain elusive, as existing models depend on FMR1 KO. To overcome this limitation we developed a novel model to investigate FMR1 silencing during neurodevelopment by generating human brain organoids starting from patient-derived naïve induced pluripotent stem cells (iPSCs). Differently from conventional primed iPSCs, naïve are hypomethylated and express FMR1. Starting this state allows us to track specific changes leading to FXS, including FMR1 silencing, which is recapitulated in our model. Moreover, we found that FMRP levels start to decrease by day 30 of differentiation in FXS organoids, which display more than 150 differential proteins when compared with healthy lines. These exponentially increase over neuronal maturation until day 90, when we see upregulation of many FMRP targets and autism-related proteins, involved in dendritic spine morphology and synaptic functions. To identify the cell types associated with the specific protein production patterns, we make use of our scRNAseq dataset to deconvolve cellular origin of upregulated proteins. We discovered a strong enrichment in mature neuronal types, such as GABAergic neurons, microglia-like cells, astrocytes and oligodendrocytes. To dissect the cell type-specific contributions to FXS pathogenesis, we combined our model with a metabolic non-canonical amino acid (ncAA) tagging strategy. We generated transgenic FXS-derived naïve hiPSCs expressing the mutant tRNAse MetRSL274G under specific neuronal or astrocyte promoters (SYN1/GFAP). The ncAA is incorporated into proteins which can be further coupled with fluorophores or affinity tags via click chemistry for direct visualization, quantification, or enrichment of the nascent proteome. We successfully validated the cell lines’ ability to differentiate into human brain organoids and to label proteins in specific cell-types. Overall, we established a system to gain access to a previously inaccessible time window of FXS disease. This allowed us to discover when and where FMR1 silencing takes place in a human system. We can now dissect the role of specific cell types at an early disease stage for the identification of novel markers that can be used as pharmacological targets.