MECP2 Changes Dictate Nerve Cell Structure and Function in Study
Patient with mild symptoms, patient with severe symptoms featured in study
Nerve cells derived from Rett syndrome patients showed alterations in structure, function, and network connectivity that depend on the severity of MECP2 genetic changes that cause the condition, a study revealed.
Nerve cells from a rare atypical Rett patient with mild disease and preserved speech had similar core structural changes but limited functional and network defects compared to cells derived from a severe Rett patient.
The study, “Wide spectrum of neuronal and network phenotypes in human stem cell-derived excitatory neurons with Rett syndrome-associated MECP2 mutations,” was published in the journal Translational Psychiatry.
Up to 95% of Rett syndrome cases are caused by mutations in the MECP2 gene, which encodes the protein of the same name: MECP2. This protein binds to DNA and regulates the activity of other genes by regulating the structure of chromatin, the bundle of DNA and proteins that makes up chromosomes. Such chromatin changes control the activity of other genes by switching them on or off.
Because the MECP2 protein is found mainly in nerve cells (neurons), these mutations lead to a faulty MECP2 that affects proper brain development and function. Although symptoms vary widely among Rett patients, the condition typically impairs the ability to walk and communicate.
Models of Rett neurons derived from stem cells have demonstrated many cellular features seen in mouse models and Rett patients.
Mutant MECP2 protein with a reduced ability to bind to DNA
In this work, researchers in Canada, along with collaborators in the U.S., compared stem cell-derived neurons from a patient with classic severe Rett-causing mutations to those derived from an atypical Rett patient with milder disease and preserved speech.
This patient carries a MECP2 mutation that resulted in a mutant MECP2 protein (L124W) with a reduced ability to bind to DNA. Symptoms include typical hand stereotypies (repetitive movements) but good hand skills, normal head size, language recovery using single words or phrases, and milder intellectual disabilities.
Initial experiments demonstrated that chromatin was disrupted when mouse cells carried one of two MECP2 mutations associated with severe Rett. In contrast, in cells with MECP2-L124W, there was limited chromatin disruption. Experiments confirmed the MECP2-L124W protein had reduced DNA binding ability.
The team created stem cell-derived neurons from the L124W patient and a girl with severe Rett who carries a rare null mutation in which MECP2 was completely absent. Two other controls’ cell lines were generated by deleting MECP2. Experiments showed that MECP2-L124W mutant protein was present and produced at unchanged levels in L124W cells.
Neuron function was assessed using whole-cell patch clamp, a method to study the flow of ions in and out of neurons, which generates electrical currents.
Alterations in current flow were seen in neurons without MECP2, as well as a deficiency in their action potential firing, or the ability to stimulate an electrical signal. The amplitude and frequency of impulses were also reduced in these cells, similar to known Rett-associated characteristics, the team noted.
In comparison, while currents in L124W-patient neurons were smaller than those in control cells, these neurons did not display a deficiency in firing action potentials nor reductions in impulse amplitude or frequency.
As expected, imaging analysis showed neurons lacking MECP2 were smaller with reduced dendrite length — branches extending from nerve cell bodies that connect with nerve fibers (axons) from other neurons to communicate. In contrast, the L124W neurons showed no changes in cell body area or dendrite length.
MECP2-deleted cells also had a lower density of synapses, the signaling gap between axons and dendrites, but L124W neurons did not. Closer analysis showed L124W neurons had decreased branching complexity relative to controls, similar to the reduced branching complexity in MECP2-deleted neurons.
Overall, “L124W neurons show only the core subset of dendrite complexity changes shared with MECP2 null neurons,” the researchers noted.
During brain development, individual neurons connect to form networks that produce distinct patterns of synchronized electrical activity. Such patterns help shape the network structure and function in the developing brain.
High-frequency neural activity
All neuron samples were co-cultured with mouse astrocytes, cells of the nervous system that support neurons and facilitate network formation. After three weeks, current recordings were collected weekly, showing the emergence of high-frequency neural activity.
Neuron networks derived from the L124W patient did not show significant differences in mean electrical impulse (firing) rate compared to normal controls. At later time points, the frequency of electrical bursts throughout the network increased, and their duration decreased, but no other differences were observed.
Comparatively, MECP2-delete network electrical bursts showed a consistent decrease in frequency relative to controls beginning at four weeks, followed by longer network burst durations that emerged at weeks five–six.
“We used a multilevel approach to identify a range of [Rett]-associated [characteristics] relating to form and function in isogenic human stem cell-derived excitatory neurons harbouring a novel [L124W] or MECP2 null mutations,” the authors wrote. “We define a core set of cellular and network phenotypes in atypical [Rett] neurons, and identify additional synaptic and morphological phenotypes present in null neurons.”
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