Generation and Characterization of a Skeletal Muscle Cell-Based Model Carrying One Single Gne Allele: Implications in Actin Dynamics

Abstract

UDP-N-Acetyl glucosamine-2 epimerase/N-acetyl mannosamine kinase (GNE) catalyzes key enzymatic reactions in the biosynthesis of sialic acid. Mutation in GNE gene causes GNE myopathy (GNEM) characterized by adult-onset muscle weakness and degeneration. However, recent studies propose alternate roles of GNE in other cellular processes beside sialic acid biosynthesis, particularly interaction of GNE with α-actinin 1 and 2. Lack of appropriate model system limits drug and treatment options for GNEM as GNE knockout was found to be embryonically lethal. In the present study, we have generated L6 rat skeletal muscle myoblast cell-based model system carrying one single Gne allele where GNE gene is knocked out at exon-3 using AAV mediated SEPT homology recombination (SKM-GNEHz). The cell line was heterozygous for GNE gene with one wild type and one truncated allele as confirmed by sequencing. The phenotype showed reduced GNE epimerase activity with little reduction in sialic acid content. In addition, the heterozygous GNE knockout cells revealed altered cytoskeletal organization with disrupted actin filament. Further, we observed increased levels of RhoA leading to reduced cofilin activity and causing reduced F-actin polymerization. The disturbed signaling cascade resulted in reduced migration of SKM-GNEHz cells. Our study indicates possible role of GNE in regulating actin dynamics and cell migration of skeletal muscle cell. The skeletal muscle cell-based system offers great potential in understanding pathomechanism and target identification for GNEM.

Keywords: Actin dynamics; GNE myopathy; L6 myoblast; SEPT homology recombination; Sialylation; Single gene mutation disorders.