Background and objectives Sporadic inclusion body myositis (IBM) is the most common form of myositis. The underlying cause of the disease is still unknown. Recently, the enzyme cytosolic 5’-nucleotidase 1A (cN-1A) was found to be a frequent target of serum autoantibodies in IBM but not other forms of myositis or other neuromuscular and inflammatory diseases.¹ cN-1A, a member of the 5’-nucleotidase family, is known to catalyse the conversion of adenosine monophosphate to adenosine and phosphate but its subcellular localisation has not been well investigated. We aimed to characterise cN-1A distribution and localisation both in a variety of cell lines and in skeletal muscle tissues from patients with various forms of myositis and healthy controls.

Materials and methods We investigated the cN-1A expression in various cell lines, both in untransfected cells and in cells transfected with cN-1A constructs encoding GFP-tagged or non-tagged cN-1A (mutant) protein: 1) pEGFP-cN-1A; 2) pcDNA4-cN-1A and 3) pcDNA4-cN-1A[D211A], a point mutant predicted to abolish cN-1A activity. A polyclonal anti-cN-1A antibody was used to visualise the non-tagged protein. We investigated cN-1A expression and localisation in skeletal muscle tissue from 8 IBM, 8 PM and 6 DM patients and compared this to healthy skeletal muscle tissue and tissues from other diseases.

Results Endogenous cN-1A expression in acetone:methanol-fixed cell lines showed a diffuse cytosolic staining pattern. When GFP-tagged cN-1A was overexpressed we observed structures resembling, but not the same as, previously described ‘Rods and Rings’.² Intense accumulations of cN-1A in the cytoplasm and/or close to the nuclear membrane were also observed, particularly in cells overexpressing non-tagged cN-1A, and not cells expressing cN-1A[D211A]. In all skeletal muscle tissues, we observed a checkerboard pattern of cN-1A immunostaining which was more intense in type II fibres than type I fibres. We also observed perinuclear staining in all samples and localisation of cN-1A close to the Z-line in longitudinal fibres. In the skeletal muscle of IBM patients we found strongest cN-1A staining in small-sized and/or atrophied (type II) fibres and co-localisation of cN-1A with p62 in rimmed vacuoles. In fibres containing rimmed vacuoles, cN-1A was not restricted to co-localisation with p62.

Conclusions Our cell culture experiments show that increased expression of active cN-1A alters its distribution and leads to accumulation of cN-1A in various cell lines. Together with our immunochemical findings, our results suggest that altered expression and accumulation of cN-1A could be of physiological relevance in IBM and requires further investigation.

References

1. Pluk H, van Hoeve BJA, van Dooren SHJ, et al. Autoantibodies to cytosolic 5′-nucleotidase 1A in inclusion body myositis. Ann Neurol 2013; 73 :397–407

2. Carcamo WC, Satoh M, Kasahara H, et al. Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells. PLoS One 2011; 6 :e29690