Compromised membrane repair in muscle cells from GNE myopathy patients

Abstract

GNE myopathy (GNEM) is an adult onset autosomal recessive neuromuscular disease that initially presents as distal muscle weakness and slowly progresses to atrophy of skeletal muscles throughout the body. GNEM results from mutations in the GNE gene that is located on chromosome 9 and encodes for the N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase (GNE) which is the rate‐limiting enzyme for the biosynthesis of sialic acid. Sialic acid plays an important role in cellular interactions through modification of the terminal carbohydrate chains on glycoproteins in the plasma membrane. While minimal GNE activity is required for viability, hyposialylation has been observed in GNEM patients. Even though the mechanism of the disease requires further investigations beyond the impaired sialic acid pathway, hyposialylation is still thought to be responsible, at least partially, for the disease pathology. This study aims to elucidate the molecular mechanism of GNEM that links hyposialylation to muscle atrophy and weakness. The association of sialic acid with membrane proteins suggests possible implications for the plasma membrane integrity and repair capacity. We hypothesized that GNEM involves reduced membrane repair capacity in skeletal muscle. To test this hypothesis, we performed laser injury assays on myoblasts transdifferentiated from human skin fibroblasts of GNE patients. Isolated fibroblasts were transdifferentiated into myoblasts using overexpression of MyoD. A multiphoton laser was used to injure the myoblasts in the presence of FM4‐64 lipophilic dye. The resealing capacity is measured by observing the change in fluorescent dye uptake following injury. Our results indicate reduced sarcolemmal membrane repair in GNE myoblasts when compared to healthy controls. To test if sialic acid is required for effective membrane repair, we assessed the level of plasma membrane damage using a glass bead wounding assay on C2C12 myoblasts treated with neuraminidase to remove sialic acid from these cells. The results indicate that sialic acid depleted myoblasts have a more fragile plasma membrane. To further elucidate the mechanism of GNEM we measured the expression levels of key membrane repair proteins in myoblasts and myotubes derived from GNE patients and healthy volunteers. Overall, our results indicate that hyposialylation is correlated with reduced sarcolemmal membrane integrity and repair capacity. To confirm this as a mechanism of GNE myopathy, future studies will use shRNA to knock down GNE in electroporated wild‐type mouse muscle. Muscles from the electroporated mice will be tested using laser injury and western blots to confirm the results we observed in vitro. Future studies also include analyzing expression levels of sarcolemma repair proteins in GNEM patients. Preliminary data from our studies show that sialic acid deficiency is associated with impaired sarcolemma repair leading to muscle atrophy in GNEM.