Summary Rett Syndrome (RTT) is caused by mutations of MECP2, a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show, using an isogenic human embryonic stem cell model of RTT, that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as global gene activator in neurons but not in neural precursors. Decreased transcription in neurons was coupled with a significant reduction in nascent protein synthesis and lack of MECP2 was manifested as a severe defect in the activity of the AKT/mTOR pathway. Lack of MECP2 also leads to impaired mitochondrial function in mutant neurons. Activation of AKT/mTOR signaling by exogenous growth factors or by depleting PTEN boosted protein synthesis and ameliorated disease phenotypes in mutant neurons. Our findings indicate a vital function for MECP2 in maintaining active gene transcription in human neuronal cells.

Rett Syndrome (RTT) is an X-linked, neurodevelopmental disorder caused primarily by mutations in the Methyl-CpG-binding protein 2 (MECP2) gene, which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. While postnatal functions of MeCP2 have been thoroughly investigated, its role in prenatal brain development remains poorly understood. Given the well-established importance of miRNAs in neurogenesis, we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 shRNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs, we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK/MAPK) and protein kinase B (PKB/AKT) signaling. In parallel, we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and 3D (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors (NPs) deficient in MeCP2 restored AKT and ERK activation, respectively, and ameliorated the observed alterations in neuronal differentiation. Moreover, overexpression of miR-199 or miR-214 in WT mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together, our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders.

Rett Syndrome was long considered to be simply a disorder of postnatal development, with phenotypes that manifest only late in development and into adulthood. A variety of recent evidence demonstrates that the phenotypes of Rett Syndrome are present at the earliest stages of brain development, including developmental stages that define neurogenesis, migration, and patterning in addition to stages of synaptic and circuit development and plasticity. These phenotypes arise from the pleotropic effects of MeCP2, which is expressed very early in neuronal progenitors and continues to be expressed into adulthood. The effects of MeCP2 are mediated by diverse signaling, transcriptional, and epigenetic mechanisms. Attempts to reverse the effects of Rett Syndrome need to take into account the developmental dynamics and temporal impact of MeCP2 loss.

Somatic hypermutation (SHM), coupled with Ag selection, provides a mechanism for generating Abs with high affinity for invading pathogens. Class-switch recombination (CSR) ensures that these Abs attain pathogen-appropriate effector functions. Although the enzyme critical to both processes, activation-induced cytidine deaminase, has been identified, it remains unclear which cis-elements within the Ig loci are responsible for recruiting activation-induced cytidine deaminase and promoting its activity. Studies showed that Ig gene-transcription levels are positively correlated with the frequency of SHM and CSR, making the intronic, transcriptional enhancer Eμ a likely contributor to both processes. Tests of this hypothesis yielded mixed results arising, in part, from the difficulty in studying B cell function in mice devoid of Eμ. In Eμ’s absence, VH gene assembly is dramatically impaired, arresting B cell development. The current study circumvented this problem by modifying the murine Igh locus through simultaneous insertion of a fully assembled VH gene and deletion of Eμ. The behavior of this allele was compared with that of a matched allele carrying the same VH gene but with Eμ intact. Although IgH transcription was as great or greater on the Eμ-deficient allele, CSR and SHM were consistently, but modestly, reduced relative to the allele in which Eμ remained intact. We conclude that Eμ contributes to, but is not essential for, these complex processes and that its contribution is not as a transcriptional enhancer but, rather, is at the level of recruitment and/or activation of the SHM/CSR machinery.