Combined MeCP2 changes may help explain varied Rett symptoms

Modifications to MeCP2, the protein whose dysfunction underlies most cases of Rett syndrome, may help explain the wide range of symptoms seen in people with the condition, a new mouse study suggests.

The study found that combining two chemical MeCP2 modifications produced complex behavioral and molecular effects that differed from patterns previously reported for either modification alone. The findings offer a more detailed picture of how the protein regulates brain function and may help guide future research.

The study, “Single-cell multiome profiling reveals neuronal bias of modulatory role of MeCP2 phosphorylation,” was published in Neurobiology of Disease.

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How MeCP2 changes may affect brain function

MeCP2 is a protein that helps control gene activity in cells, particularly in the brain. Mutations in the MECP2 gene, which provides instructions for making the protein, cause most cases of Rett syndrome. The disorder is marked by cognitive difficulties, movement problems, seizures, and repetitive hand movements.

MeCP2 is important for the normal function of neurons, or nerve cells, including those in the hippocampus, a brain region involved in learning and memory. It’s thought that MeCP2 deficiency disrupts the balance between excitatory neurons, or “go” signals, and inhibitory neurons, or “stop” signals, a balance important for normal learning and memory.

After it is made, the MeCP2 protein can undergo a chemical modification called phosphorylation, which can change its activity in response to cellular signals.

Two of the most studied MeCP2 phosphorylation sites are called serine 421, or S421, and serine 80, or S80. S421 becomes phosphorylated when neurons are active, while S80 is phosphorylated in resting neurons and becomes dephosphorylated when neurons are activated.

Each site had previously been studied independently, but researchers at the University of Wisconsin-Madison examined what happened when both were changed at the same time.

The researchers developed a mouse line carrying both mutations, creating a fixed phosphorylation pattern resembling the one seen in active neurons. S421 was changed to mimic phosphorylation, while S80 was changed to prevent it.

The mice first completed a series of behavioral tests. In a running-wheel test, mutant mice ran less than wild-type mice. In an open-field test, in which mice could move freely in an enclosed space, mutant mice traveled a shorter total distance and reared up on their hind legs less often, also indicating reduced movement.

In a fear-conditioning test, in which mice learned to associate a place with a mild shock, mutant mice froze more than wild-type mice when placed back in the same setting, suggesting a stronger memory of where the shock occurred. No differences were found between the groups in tests of social behavior or motor learning.

Combined mutations produce distinct behavioral patterns

When the findings were compared with previously published data on mice carrying only the S80 or only the S421 mutation, three different patterns emerged. For the running-wheel test, mice with either single mutation or both mutations showed reduced activity or a trend toward reduced activity, suggesting that combining the mutations did not appear to further reduce activity beyond either mutation alone.

“This pattern suggests that for this particular function, S80 and S421 phosphorylation may work synergistically,” the team noted.

In the open-field test, neither single mutation had previously shown an effect, but the double mutation led to increased exploration of the center of the enclosure. In fear conditioning, the double mutant showed the opposite pattern from either single mutant: mice with either single mutation had shown a weaker memory of the setting where the shock occurred, while double-mutant mice showed a stronger memory of that setting.

The researchers also measured the electrical activity of neurons in the hippocampus. Experiments found no differences in the neurons’ basic properties between mutant and wild-type mice. However, spontaneous excitatory signals were more frequent and had a greater amplitude, or strength, in double-mutant mice than in wild-type mice, suggesting increased excitatory signaling between neurons.

“These electrophysiological changes are consistent with the observed behavioral phenotypes [characteristics],” the team noted.

To understand what might be driving these behavioral changes, the team analyzed gene activity in individual cells from the hippocampus. The effects of the mutations were concentrated mainly in neurons, particularly excitatory neurons, rather than spread evenly across all hippocampal cell types.

To confirm these results, researchers used a second technique called MERFISH, which allowed gene activity to be measured at specific locations within tissue. By examining 300 selected genes in hippocampal tissue, they found that most gene-activity changes among excitatory neurons occurred in CA1, one of four hippocampal subregions examined and a major output region of the hippocampus.

“These findings advance our understanding of how combinatorial post-[protein-production] modifications tune MeCP2 function and highlight the importance of studying multi-site regulation in neurodevelopmental disorders,” the team wrote.