Fragile X Syndrome (FXS) is an X-linked neurodevelopmental disorder and the main form of inherited intellectual disability. It is caused by the expansion of a CGG sequence to more than 200 repeats in the promoter of the fragile X mental retardation 1 gene (FMR1) that determines its silencing through methylation during embryonic development. Evidence from chorionic villi analysis reports that the full mutated (FM) FMR1 gene is actively expressed during the first trimester of gestation, but the exact time and process of silencing are still unclear.
In this context, our work aims to develop a more accurate in vitro model of the developing FXS human brain in which to define the dynamics of FMR1 gene silencing by identifying the time and cell population in which its expression is switched off.
To follow our purpose, we developed an innovative in vitro model of human embryonic neurodevelopment generated from naïve induced pluripotent stem cells (iPCSs).
Naïve iPSCs are characterized by a broad hypomethylated genome, including in the FMR1 locus, and better resemble what happens in vivo compared to conventional primed hypermethylated iPSCs in which the FMR1 locus is not reactivated upon reprogramming.
Thus, we developed a highly efficient reprogramming method via microfluidics that allowed us to reprogram FXS fibroblasts into primed and isogenic naïve iPSCs. We confirmed the unique ability of naïve iPSCs to demethylate the FMR1 promoter, reactivating its transcription and translation.
Then, we set up a differentiation protocol for naïve iPSCs into 3D cortical brain organoids, which mimics the human cortical brain development. We applied it on FM demethylated naïve FXS iPSCs, to investigate FMR1 expression through differentiation and maturation.
We followed the neural differentiation of FXS cortical organoids for up to 4 months and we observed either that: i) hypermethylation of FMR1 locus occurred very rapidly during neural differentiation and both FMR1 mRNA and protein were downregulated; ii) FMR1 locus methylated only partially (in longer alleles) and the unmethylated alleles underwent shortening falling in the premutation category; iii) FMR1 locus did not methylate leading to an increased expression of FMR1 mRNA more than 5 folds compared to the healthy control associated to the presence of intranuclear inclusions, typical of permutations.
The results of our work show that we have a tractable model in which we can investigate key targetable mechanisms of FXS pathogenesis. In our 3D FXS cortical organoids we can identify which cell type is more sensitive to FMR1 silencing, helping the development of suitable treatments for FXS patients.
Organoidi corticali come modello per lo studio dell'espressione di FMR1 durante le prime fasi di neurosviluppo in pazienti della sindrome dell'X fragile
La sindrome dell'X fragile è la principale causa monogenica di autismo e la più frequente forma di disabilità mentale. È causata dall'espansione trinucleotidica di CGG nel promotore del gene FMR1 e dal successivo silenziamento dello stesso. Da analisi condotte su villi corionici, la proteina FMRP è presente durante il primo trimestre di gestazione, dopodiché risulta assente.
L'obiettivo del nostro progetto è investigare le modifiche genetiche ad epigenetiche del gene FMR1 alla base della patologia durante lo sviluppo neuroembrionale. Per raggiungere il nostro obiettivo, abbiamo generato un modello in vitro paziente specifico dello sviluppo della corteccia partendo da cellule staminali. Tramite la riprogrammazione di cellule somatiche di paziente a cellule staminali indotte a uno stadio naïve, ovvero corrispondente a uno stadio pre-impianto dell'embrione, è possibile riattivare l'espressione di FMR1. In seguito, queste cellule staminali indotte sono state differenziate in organoidi corticali, che ricapitolano in vitro lo sviluppo neuroembrionale che avviene in vivo.
Lo studio delle primissime fasi embrionali di neurodifferenziamento tramite gli organoidi derivati da cellule di paziente può portare all'individuazione di meccanismi chiave per la patogenesi della sindrome dell'X Fragile e al conseguente sviluppo di strategie terapeutiche.