Generation and Characterization of a Human Neuronal In Vitro Model for Rett Syndrome Using a Direct Reprogramming Method

Rett Syndrome (RTT) is a severe neurodevelopmental disorder, afflicting 1 in 10,000 female births. It is caused by mutations in the X-linked methyl-CpG-binding protein gene (MECP2), which encodes for the global transcriptional regulator methyl CpG binding protein 2 (MeCP2). As human brain samples of RTT patients are scarce and cannot be used for downstream studies, there is a pressing need for in vitro modeling of pathological neuronal changes. In this study, we use a direct reprogramming method for the generation of neuronal cells from MeCP2-deficient and wild-type human dermal fibroblasts using two episomal plasmids encoding the transcription factors SOX2 and PAX6. We demonstrated that the obtained neurons exhibit a typical neuronal morphology and express the appropriate marker proteins. RNA-sequencing confirmed neuronal identity of the obtained MeCP2-deficient and wild-type neurons. Furthermore, these MeCP2-deficient neurons reflect the pathophysiology of RTT in vitro, with diminished dendritic arborization and hyperacetylation of histone H3 and H4. Treatment with MeCP2, tethered to the cell penetrating peptide TAT, ameliorated hyperacetylation of H4K16 in MeCP2-deficient neurons, which strengthens the RTT relevance of this cell model. We generated a neuronal model based on direct reprogramming derived from patient fibroblasts, providing a powerful tool to study disease mechanisms and investigating novel treatment options for RTT.