A2.4 Subcellular localisation of cytosolic 5’-nucleotidase 1A in cell lines and skeletal muscle: implications for inclusion body myositis
Authors: M Herbert, C Onnekink, JJ Engels, B Küsters, B van Engelen, G Pruijn
Journal: Annals of the Rheumatic Diseases (2015)

<h3>Background and objectives</h3> 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.<sup>1</sup> 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. <h3>Materials and methods</h3> 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. <h3>Results</h3> 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’.<sup>2</sup> 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. <h3>Conclusions</h3> 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. <h3>References</h3> Pluk H, van Hoeve BJA, van Dooren SHJ, <i>et al</i>. Autoantibodies to cytosolic 5′-nucleotidase 1A in inclusion body myositis. <i> Ann Neurol </i> 2013; <b> 73 </b>:397–407 Carcamo WC, Satoh M, Kasahara H, <i>et al</i>. Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells. <i> PLoS One </i> 2011; <b> 6 </b>:e29690