Wide spectrum of neuronal and network phenotypes in human stem cell-derived excitatory neurons with Rett syndrome-associated MECP2 mutations

Abstract

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by heterozygous loss-of-function mutations in the X-linked gene MECP2 that is a global transcriptional regulator. Mutations in the methyl-CpG binding domain (MBD) of MECP2 disrupt its interaction with methylated DNA. Here, we investigate the effect of a novel MECP2 L124W missense mutation in the MBD of an atypical RTT patient with preserved speech in comparison to severe MECP2 null mutations. L124W protein had a limited ability to disrupt heterochromatic chromocenters due to decreased binding dynamics. We isolated two pairs of isogenic WT and L124W induced pluripotent stem cells. L124W induced excitatory neurons expressed stable protein, exhibited increased input resistance and decreased voltage-gated Na+ and K+ currents, and their neuronal dysmorphology was limited to decreased dendritic complexity. Three isogenic pairs of MECP2 null neurons had the expected more extreme morphological and electrophysiological phenotypes. We examined development and maturation of L124W and MECP2 null excitatory neural network activity using micro-electrode arrays. Relative to isogenic controls, L124W neurons had an increase in synchronous network burst frequency, in contrast to MECP2 null neurons that suffered a significant decrease in synchronous network burst frequency and a transient extension of network burst duration. A biologically motivated computational neural network model shows the observed changes in network dynamics are explained by changes in intrinsic Na+ and K+ currents in individual neurons. Our multilevel results demonstrate that RTT excitatory neurons show a wide spectrum of morphological, electrophysiological and circuitry phenotypes that are dependent on the severity of the MECP2 mutation.

Hyperexcitability in humanMECP2null neuronal networks manifests as calcium-dependent reverberating super bursts

“…CLT iPSC lines were derived from an atypical RTT patient with the milder phenotype of the preserved speech variant (heterozygous L124W missense mutation) and express only the mutant or the wildtype allele due to X-chromosome inactivation. Stem cell-derived MECP2 null excitatory neurons from WIBR3 and PGPC14 have smaller cell bodies and dendritic arbours relative to isogenic controls, but CLT neurons have very minor changes in dendritic branching only 7 .…”

“…In brief, Mok et al (2022) generated excitatory neurons from three isogenic pairs of female pluripotent stem cell lines (WIBR3, PGPC14, and CLT) using the Ngn2 protocol (fig. 1, b and 7 ). The MECP2 null pairs were derived by gene editing of the WIBR3 human embryonic stem cell (ESC) line or the PGPC14 healthy induced pluripotent stem cell (iPSC) line.…”

“…Electrophysiological data recorded by Mok et al (2022) from MECP2 mutant excitatory neurons and neuronal networks including their isogenic controls were obtained from NDD-Ephys-dB (see Materials and Methods and 7 ). This newly created database facilitates sharing of raw data from electrophysiology experiments and incorporates a toolbox for improved exploration and visualization of results.…”

“…Supporting this hypothesis, rodent models show delayed maturation of functional synaptic connections 4,24 . Additionally, we previously reported that human stem cell-derived excitatory neurons harbouring an MECP2 null mutation produce morphologically and functionally hypo-connected neurons with reduced spontaneous action potential numbers 7 . The same MECP2 null neurons, when isolated from the network, showed changes in intrinsic properties such as increased input resistance which is generally inversely related to rheobase, suggesting hyperexcitable neurons 7 .…”

“…Additionally, we previously reported that human stem cell-derived excitatory neurons harbouring an MECP2 null mutation produce morphologically and functionally hypo-connected neurons with reduced spontaneous action potential numbers 7 . The same MECP2 null neurons, when isolated from the network, showed changes in intrinsic properties such as increased input resistance which is generally inversely related to rheobase, suggesting hyperexcitable neurons 7 . Together with decreased capacitance 7 , input resistance jointly contributes to the neuron’s time course and response to current injections.…”