Elucidating nebulin expression and function in health and disease

The nemaline myopathies (NM) are a group of genetic muscle disorders, ranging in severity from severe, early lethal forms to milder muscle disorders with onset in childhood, sometimes presenting in adulthood. Nemaline myopathies present with usually non-progressive or slowly progressive generalised muscle weakness and are pathologically characterised by the presence of cytoplasmic inclusions called nemaline bodies or rods. So far, mutations in twelve genes have been identified underlying the disorder. This Thesis aimed at elucidating the expression of the nebulin gene (NEB) and the function of the protein, in order to gain insight into the pathogenetic mechanisms leading to NM. Approximately half of NM cases are caused by mutations in NEB, and the vast majority of these are inherited recessively. NEB consists of 183 exons, giving rise to transcripts up to 26 kb in length. Nebulin is an enormous protein, with important roles in maintaining the structure and function of skeletal muscle. It is localised in the thin filament of the muscle sarcomere, and one nebulin protein spans the entire length of the filament. Nebulin structure is highly modular, consisting of actin-binding simple repeats, and super repeats formed by seven simple repeats. In NEB, more than 240 different pathogenic variants have been published, causing myopathies with no cure currently available. Furthermore, most NEB-NM patients have a compound heterozygous genotype, with two private mutations anywhere along the gene, which further hampers the discovery of any genotype-phenotype correlations. Many of the NEB exons are alternatively spliced. In this study, we investigated NEB expression at the RNA level, in 21 different skeletal muscles and in brain. We demonstrated co-expression of several different nebulin isoforms in all the muscles studied, but no isoforms specific for certain muscles were identified. The results also indicated that all 183 NEB exons are expressed in adult brain, and that the expression levels were comparable with expression in skeletal muscle. Further studies are warranted to elucidate the expression in brain. We however, went on to further investigate the expression and function of NEB in skeletal muscle. Exons 143 and 144 are mutually exclusive, encoding the super repeats S21a and S21b, respectively. The amino acid sequences of the alternative region differ in both charge and hydrophobicity. We developed specific antibodies for these two isoforms, enabling studies of their expression at the protein level. Our results showed differences in the usage of these isoforms between various myofibres, and during muscle development. S21b was the only isoform present in early myotubes, whereas regulated expression of S21a appeared later, mainly in fast myofibres. These results form a basis for studying the functional differences between the isoforms, and add to the basic knowledge of normal muscle function. The size of nebulin complicates studies at the protein level as well as genetic studies. As the superrepeat region encompasses nearly the entire protein, we constructed a panel of 26 mini-genes, corresponding to all the different nebulin super repeats, to enable protein interaction studies. The studies covering the entire nebulin super-repeat region revealed differences in actin binding between the seemingly similar super repeats, depending on their location in the protein. Actin binding was significantly stronger towards the end of the protein, while the entire central region bound actin weakly. This finding constitutes an important step in understanding nebulin function, and may facilitate the interpretation of the effects of various NEB mutations, depending on their location along the protein. Furthermore, the super-repeat panel can be utilised for studying mutational effects functionally at the protein level. To shed light on the pathogenetic mechanisms underlying NM, and to enable the study of nebulin function in normal and defective muscle, we developed the first murine model of NM with compound heterozygous Neb mutations. The aim was to produce a model recapitulating the most common form of NM, i.e. the typical congenital form. By studying the new compound heterozygous model, and the parental lines, we have gained valuable insight into the effects of Neb mutations on muscle function. The mouse models will be useful in deciphering the pathogenetic mechanisms of NEB-NM, and will be suitable for the assessment of potential therapeutic approaches. The results of this Thesis shed light on the expression and function of nebulin in health and disease. This brings us closer to understanding the pathogenetic mechanisms of NM, and how the disorder evolves, which in turn will enable providing more accurate diagnosis and prognosis for NM patients. Understanding the pathogenetic mechanisms underlying NM is also a prerequisite for developing effective therapies for NM in the future.