Mutations in the MFN2 gene are associated with altered connections between mitochondria and another cellular structure called endoplasmic reticulum (ER). These correlate with Charcot–Marie–Tooth disease type 2A (CMT2A) severity, as suggested by a study in patient-derived cells.
The study, “MFN2 mutations in Charcot–Marie–Tooth disease alter mitochondria-associated ER membrane function but do not impair bioenergetics,” was published in the journal Human Molecular Genetics.
Although nearly 100 mutations in the MFN2 gene have been associated with the disease, the relationship between genetic variants and clinical manifestations is yet to be established.
The MFN2 protein promotes fusion of mitochondria, the cell’s power plants, while also being involved in the binding of mitochondria to the ER — a key step in protein production and transport — at the mitochondria-associated ER membrane (MAM). This membrane regulates the production of the main lipid (fat) components of myelin, the protective layer of nerve fibers.
Studies of nervous tissue and cells called fibroblasts from CMT2A patients with mutations in MFN2 revealed altered mitochondrial morphology, including swelling, degeneration, and changes in their distribution in nerve fibers, or axons. However, the findings in fibroblasts were contradictory, as to whether MFN2 protein levels, mitochondrial function and their DNA content were altered.
Work in motor neurons — specialized cells that control muscle contraction — derived from a patient with CMT2A showed some of the disease-related features, although mitochondrial morphology was unchanged and several phenotypes (observable symptoms) were relatively mild.
The impact of MFN2 mutations in the communication between mitochondria and the ER and in disease progression remain unknown. To evaluate changes in MAM function, the research team from Italy, the U.K and the U.S. used fibroblasts from three patients carrying different mutations in MFN2 — R364W, M376V and W740S — and with clinical phenotypes ranging from mild to severe.
The results first showed that the mutations were heterozygous, which means they occurred in only one of the two gene copies. Levels of MFN2 were not different comparing the three patients to controls, although the average steady-state levels of MFN2 messenger RNA (generated from DNA in protein production) were two-fold higher in the patients.
Mitochondrial imaging revealed similar length, perimeter and area of mitochondria in patients and controls. However, patients 1 (more severely affected, 32 years of age, woman) and 3 (mild severity, 43 years, also a woman) showed a greater distance between the ER and mitochondria, while increased length of the mitochondria-ER contact was found in patient 1 only.
All three patients demonstrated a higher number of contacts greater than a predefined width of 40 nanometers. This was consistent with the greater mitochondria-ER distances, the team commented.
Then, the investigators found that conversion of cholesterol into cholesteryl esters (CEs) — mediated by the ACAT1 enzyme in the MAM — was increased in fibroblasts from patient one, which also demonstrated greater accumulation of lipid droplets. In turn, patients 2 (moderate impairment, 60 years, man) and 3 showed a clear trend toward increased lipid droplets, as well as reduced synthesis of phospholipids, a major component of cell membranes.
Upon depletion of intracellular stores, calcium entry into the cells was significantly increased in patients 1 and 2, but not in patient 3, while baseline calcium levels were unchanged. This suggests only moderate disruptions of MFN2-mediated calcium equilibrium in CMT2A, the researchers said.
Subsequent experiments evaluated whether MFN2 mutations in fibroblasts affected their bioenergetics, or the ability to produce energy. Of note, while phospholipid synthesis and bioenergetics are mainly mitochondrial functions, CE synthesis and lipid droplets formation are mainly ER functions.
Overall, no differences in bioenergetics were found, although the oxygen consumption rate was slightly increased in patients 2 and 3 under baseline conditions.
“Taken together, our findings could have implications for the treatment of this disorder and might even provide a conceptual basis underlying the pathogenesis of both axonal and demyelinating forms of the disease,” the scientists wrote.