The study, “Altered mitochondrial function in cells carrying a premutation or unmethylated full mutation of the FMR1 gene,” was published in the journal Human Genetics.
Fragile X is caused by the expansion of CGG repeats — a DNA segment — in the FMR1 gene, which provides instructions for making the FMRP protein. FMRP is involved in RNA metabolism and plays a key role in the formation and maturation of synapses — the sites where nerve cells communicate. C stands for cytosine and G for guanine, two of the four building blocks of DNA.
In most people, the number of CGG repeats ranges from fewer than 10 to about 40. “Full” mutation carriers typically have more than 200 CGG repeats, while premutation carriers have between 55 and 200 such repeats. While those with a premutation produce limited amounts of FMRP protein, people with full mutations lack this protein. This is due mainly due to the addition of methyl groups — a process called methylation — that work as “switch off” signals for FMRP production.
Rarely, people can carry an unmethylated full mutation (UFM) that leads to some production of FMRP. Notably, no intellectual disability is seen in such cases.
Those with a premutation may develop FXTAS. Despite the available genetic information on this disorder, scientists are still unsure of how affected individuals develop movement and cognition problems associated with neurodegeneration.
To learn more, researchers from Italy and Canada used human fibroblasts — the most common cell type of connective tissue — to analyze protein content. The fibroblasts were derived from three healthy people (controls), three patients with fragile X, and two carriers of UFM.
The assessment identified 282 proteins in control cells, 223 in fragile X cells, and 200 in UFM cells. The researchers detected higher levels of seven proteins related to the mitochondria only in UFM cells. One of these proteins was SOD2.
Besides energy production, mitochondrial metabolism is involved in the regulation of genes in the cell’s nucleus, which could help explain neurodegeneration in premutation carriers with FXTAS.
The researchers then focused on the SOD2 protein, which is an important regulator of oxidative stress. This process, seen in people with FXTAS, occurs when antioxidant defenses no longer protect against the production of reactive oxygen species (ROS). High levels of these free radicals cause damage in cell components, such as proteins and DNA, and can lead to the death of nerve cells.
Protein and RNA levels of SOD2 were three to five times higher in UFM cells compared with controls. Similar high levels were seen in cells from premutations carriers, while those with fragile X had lower levels of SOD2 than control cells.
To understand these findings, the team then investigated the potential underlying processes. No relevant differences in methylation were seen across the groups. Yet, the messenger RNA (mRNA) of SOD2 — generated from DNA in protein production — was bound to the FMRP protein at significantly higher levels in UFM cells compared with other cell types.
In addition, levels of ROS and superoxide — one of the toxic ROS species, which is targeted by the SOD2 enzyme — were reduced in UFM cells.
Next, the investigators evaluated how changes in SOD2 altered mitochondria. Although total mitochondria numbers were not too different across the groups, a greater number of mitochondria in UFM cells and those with premutation showed an abnormal morphology. Instead of an elongated and tubular shape, these mitochondria had a ring-like shape.
These atypical mitochondria also had a different localization in the cell. Specifically, they were found around the nucleus and were distributed in a disorganized manner.
To validate their results, the team used a compound called MC2791 to induce high levels of SOD2. MC2791 led to lower ROS and superoxide levels as well as mitochondrial abnormalities in control and fragile X cells. Targeting the mRNA of SOD2 and FMRP to prevent their production reverted the alterations in mitochondria and increased ROS levels in premutation and UFM cells.
“Overall, these data suggest that in PM [premutation] and UFM carriers … SOD2 overexpression helps to maintain low levels of both superoxide and ROS with signs of mitochondrial degradation,” the researchers said.
“This study highlights the importance of mitochondrial outcomes as biomarkers of neurodegeneration and as potential therapeutic targets to prevent or delay FXTAS symptoms,” they added.