Speaker
Description
Fragile X Syndrome (FXS) is a rare neurodevelopmental disorder, representing a leading cause of inherited intellectual disability and monogenic cause of autism spectrum disorders. It results from abnormal CGG repeat expansion in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene, leading to its silencing and loss of FMRP, an RNA-binding protein crucial for synaptic plasticity and dendritic spine architecture. While FMR1 methylation occurs in the first trimester, the precise timing of FMRP loss and affected cell types remain unclear. Using a 3D human cortical development model derived from naïve induced pluripotent stem cells in a hypomethylated pluripotency state, we successfully recapitulated the FMR1 epigenetic dynamics observed during the early development of FXS patients’ brains. Proteomic analysis revealed that the number of dysregulated proteins in FXS increases along differentiation, half of them are direct targets of FMRP, and 1/5 are associated with autism. Most dysregulated proteins are involved in synaptic physiology and localized postsynaptically. Deconvolution of bulk proteomic with single cell RNAseq data showed that protein alterations are mainly in neuron subtypes, astrocytes and microglia. 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 glial promoters. The ncAA is incorporated into proteins which can be further coupled with different 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. This system provides access to a previously inaccessible time window of FXS disease, allowing us to discover when and where FMR1 silencing occurs in a human model and new early-stage markers for potential pharmacological targets.
| Author(s) | Carmela Ribecco*, Martina D'Ercole, Cecilia Laterza, Elisa Cesare, Alessia Gesualdo, Maria Grazia La Barbera, Roberta Frison, Roberta Polli, Alessandra Murgia, Davide Cacchiarelli, Lucia Santorelli, Paolo Grumati, Nicola Elvassore |
|---|---|
| Affiliation(s) | Veneto Institute of Molecular Medicine, Padova, Italy; Department of Industrial Engineering, University of Padova, Italy; Department of Biomedical Sciences, University of Padova, Italy; Department of Women’s and Child’s Health, University of Padova, Italy; Laboratory of Molecular Genetics of Neurodevelopment, Istituto di Ricerca Pediatrica, Città della Speranza, Padova, Italy; Department of Translational Medical Sciences, Federico II University, Naples, Italy; Department of Clinical Medicine and Surgery, Federico II University, Naples, Italy; Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy. |
