Localization of O-GlcNAc-modified proteins in neuromuscular diseases.

O-linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification of nucleocytoplasmic proteins that induces the attachment of N-acetylglucosamine to serine or threonine residues of a protein. In contrast to other protein glycosylations, this modification is highly reversible and, similar to phosphorylation, it plays important roles in various cell signals. Here, we immunolocalized O-GlcNAc-modified proteins in muscle biopsy specimens from 40 patients with neuromuscular diseases and controls. In normal muscle fibers, O-GlcNAc was found along plasma membranes and in nuclei. Diffuse and increased cytoplasmic staining of O-GlcNAc was detected in (1) regenerating muscle fibers in muscular dystrophy, myositis, and rhabdomyolysis; (2) a proportion of atrophic fibers in myositis, such as those found in perifascicular regions in dermatomyositis; and (3) vacuolated fibers in sporadic inclusion body myositis (s-IBM) and distal myopathy with rimmed vacuoles (DMRV). Target formations in neurogenic muscular atrophy were O-GlcNAc positive. Increase of O-GlcNAc glycosylation could be associated with the stress response, as these lesions have been shown to be positive for several stress markers. Vacuolar rims in s-IBM and DMRV were sometimes sharply lined by O-GlcNAc-positive deposits, which reflects myonuclear breakdown occurring from the disease.

Myonuclear breakdown in sporadic inclusion body myositis is accompanied by DNA double strand breaks.

Rimmed vacuoles in sporadic inclusion body myositis (s-IBM) contain nuclear remnants. We sought to determine if the nuclear degeneration seen in s-IBM is associated with DNA damage. In muscle biopsy specimens from ten patients with s-IBM and 50 controls, we immunolocalized 1) phosphorylated histone H2AX (γ-H2AX), which is a sensitive immunocytochemical marker of DNA double-strand breaks and 2) DNA-PK, which is an enzyme involved in double-strand break repair. In s-IBM, vacuolar peripheries often showed strong immunoreactivity to γ-H2AX and the three components of DNA-PK (DNA-PKcs, Ku70, and Ku80). A triple fluorescence study of Ku70, emerin, and DNA displayed nuclear breakdown and it suggested impaired nuclear incorporation of Ku70. The percentage of positive nuclei for γ-H2AX was significantly higher in vacuolated fibers than non-vacuolated fibers in s-IBM, or fibers in polymyosits. We hypothesize that a dysfunction of nuclear envelope may cause nuclear fragility, double-strand breaks and impaired nuclear transport in s-IBM.