Abstract: Rett syndrome (RETT) is a rare neurodevelopmental disorder, with an estimated prevalence of 1 in 10000, mostly affecting females. RETT is linked to sporadic mutations in the neurodevelopment-relevant gene Methyl-CpG-binding protein 2 (MECP2) on the X chromosome. RETT is characterized by an early period of apparently normal development, which is then followed by a sudden loss of acquired psychomotor skills. There is currently no cure for RETT. Emerging treatment strategies aim to target MECP2 expression levels and their downstream pathways. To better understand how mutations in the MECP2 gene relate to neurodevelopmental impairment, neuronal function, and the observed range of symptoms in patients, we set out to develop an integrated multimodal phenotypic screening approach. Human induced pluripotent stem cell (hiPSC)-derived neural cultures were derived from control/MeCP2-mutant lines and assessed in 2D and 3D culture systems. Cellular phenotypes were identified using a combination of functional and molecular assays, including high-throughput high-density microelectrode array (HD-MEA) measurements and gene and protein expression analyses. Preliminary data showed that MeCP2-deficient cultures exhibited higher levels of the mature neuronal marker microtubule-associated protein 2 (Map2), as well as early hyperexcitability in comparison to its WT counterpart, which may indicate MeCP2-linked neuronal differentiation deficiencies. Network activity improvement and better long-term maintenance of the 2D cultures in the presence of rat astrocytes evidenced the significance of glial cells in the context of this neurodevelopmental disorder. Neuronal networks developed more robustly in 3D-spheroid cultures with higher expression of Map2 in WT-MeCP2 cultures and spontaneously appearing astrocytes during differentiation. Further work is aimed at genetically modulating MeCP2 expression in MeCP2-mutant cultures and to assess its effect on neural culture development and electrophysiological characteristics.
