Regulatory RNAs Appear To Be Altered in Rett Models, Study Finds

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by Steve Bryson, PhD |

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Regulatory RNAs involved in nerve cell signaling and structure were altered in cell and mouse models of Rett syndrome, with these changes also evident in tissue from deceased patients, a study reported.

These findings aid in understanding the biological processes impaired in Rett and may reveal potential biomarkers to help diagnose, monitor, and treat the condition, the scientists noted.

The study, “Analysis of the circRNA and T-UCR populations identifies convergent pathways in mouse and human models of Rett syndrome,” was published in the journal Molecular Therapy Nucleic Acids.

Mutations in the MECP2 gene cause nearly 95% of Rett syndrome cases. As a result, cells are deficient in the encoded MeCP2 protein, which controls the activity of other genes by switching them on or off, and is essential in brain development and function.

A lack of MeCP2 protein has downstream consequences on multiple biological pathways, ultimately leading to characteristic Rett symptoms that include slowed growth, a loss of motor control, as well as repetitive motions, difficulty walking, and seizures.

Emerging evidence indicates that MeCP2 deficiency affects processes involving various types of RNA molecules, all of which play a central regulatory role in protein production.

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Messenger RNA (mRNA) carries instructions from genes and acts as a template to make proteins, while microRNAs (miRNA) are small segments of RNA that silence mRNA. Circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) are thought to interact and control miRNAs.

Although several studies have investigated the role of miRNAs in Rett, the consequence of MeCP2 protein deficiency on other types of RNA, such as circRNAs and lncRNAs, remains unexplored.

An international team of scientists used mouse and cell-based models of Rett, along with tissue isolated from Rett patients, to examine how this deficiency affects circRNAs and a type of lncRNA called transcribed-ultraconserved region (T-UCR), both of which are enriched in the brain.

“The lack of effective therapies and biomarkers available warrants further examination of the cellular programs underlying the disorder,” the scientists wrote.

Using mice lacking the Mecp2 gene (the mouse version of MECP2), circRNA expression (activity) profiles were examined in two brain areas implicated in Rett — the hippocampus and the frontal cortex — and compared the findings to healthy littermates.

In the hippocampus, 141 circRNAs showed increased expression (upregulated), and 247 were active at a lower level than controls (downregulated). There were fewer differences in the frontal cortex, with 47 upregulated circRNAs and 103 downregulated.

Enrichment analysis showed that circRNAs with a change of twofold or greater were derived from genes related to synapses, the sites where nerve cells communicate, and in particular, to protein receptors within synapses that bind glutamate — a chemical messenger that activates other nerve cells and a marker for Rett development.

The cytoskeleton is a dynamic network of interlinking tube-like proteins (microtubules) that helps maintain the shape and internal organization of cells.

Experiments showed a decrease in the circRNA related to SIRT2 in the frontal cortex of pre-symptomatic mice. SIRT2 is a protein thought to play a role in regulating the main protein in microtubules called alpha-tubulin.

A marked increase in SIRT2 protein was also seen in the frontal cortex (but not hippocampus) of symptomatic mice, along with a lower chemical modification of SIRT2’s primary target, alpha-tubulin. These findings were confirmed using mouse- and human-based cell models, modified to suppress MeCP2 expression.

Notably, overexpression of SIRT2-related circRNAs in cells lacking MeCP2 resulted in a significant decrease in the levels of GluR-1, GluR -2, and GluR -3, proteins that are part of glutamate receptors known as AMPAR. GluR’s drop was reversed by overexpressing MeCP2, “indicating that varying levels of circRNAs in the background of MeCP2 deficiency may contribute greatly to AMPAR biogenesis [production],” the researchers wrote.

T-UCRs are another type of RNA derived from regions in the genome that are 100% identical between humans, mice, and rats, signifying their importance in biology. In mice lacking Mecp2, alterations were found in several T-UCRs. T-UCRs were enriched in the frontal cortex, as expected, but they were also increased in the hippocampus related to processes involving AMPA receptors.

Next, the team analyzed the overall protein content within tissue from the hippocampus taken postmortem from four people without Rett and four Rett patients. Proteins significantly altered in Rett were related to microtubules as well as those associated with the activation of microglia, the primary immune cell in the brain and spinal cord.

Changes to several cytoskeleton-related proteins in the hippocampus of patients were found, including the upregulation of microtubule-associated proteins, “which might contribute to microtubule destabilization,” the scientists wrote.

Furthermore, GluR-1, GluR -2, and GluR -3 were all downregulated in the frontal cortex of symptomatic mice lacking MeCP2 and in the hippocampus of the Rett patients. Although GluR-3 protein levels were lower in Rett brain tissues, it was not accompanied by a decrease in total mRNA level, but rather the upregulation of two specific T-UCRs — uc.478 and, to a lesser extent, uc.479.

Overexpressing MeCP2 increased the expression of both GluR-3 protein and its mRNA, confirming the regulation of the gene that encodes GluR-3 (GRIA3) by MeCP2.

Finally, in MeCP2-deficient cells, a decreased was seen in the size of neurospheres — clusters of nerve stem cells grown in a lab to investigate nerve cell growth and development. Overexpressing uc.478 or uc.479 on their own restored sphere diameter in Rett cells while not affecting healthy cells, and resulted in a marked decrease in nerve cell complexity.

Consistently, when nerve stem cells differentiate into mature nerve cells, glutamate transporters associated with synapses were downregulated with overexpressed T-UCRs. “This suggests a broad effect of ultraconserved transcripts on neural stem cell physiology in the context of [Rett],” the investigators wrote.

“We detected MeCP2-dependent dysregulation of noncoding transcripts of the circRNA and T-UCR type that reveal complex regulation of … mechanisms that converge toward alterations in trafficking and glutamate receptor biogenesis,” the team wrote. “Our findings indicate that noncoding transcripts may contribute to key alterations in Rett syndrome and are not only useful tools for revealing dysregulated processes but also molecules of biomarker value.”