MODELLING SARCOGLYCANOPATHIES IN ZEBRAFISH. CHARACTERIZATION OF KNOCK OUT MUTANTS FOR δ- SARCOGLYCAN AND PHARMACOLOGICAL TESTING OF NOVEL SMALL MOLECULES.

Sarcoglycanopathies (LGMDR3-6) are a family of rare genetic diseases affecting mainly the limb girdle musculature, characterized by myalgia, progressive muscle loss and cardiomyopathy. Sarcoglycanopathies are caused by mutations in four genes encoding α-, β-, γ- and δ-sarcoglycan (SG). Until now, different animal models have been generated to unravel the molecular mechanism of this pathology. In the last years, zebrafish has become one of the most trending organisms to model human diseases, therefore we decided to mimic sarcoglycanopathy in this small vertebrate. To this intent, we knocked out the β- and δ-SG genes, but surprisingly, the novel zebrafish lines showed a very mild phenotype, almost indistinguishable from the wild-type fish, at least during the embryonic phase. As a consequence, they are not a valuable model for drug screening purposes. To overcome this problem, we decided to generate a double knock out mutant for both β and δ-SG, which indeed, is characterized by a recognizable dystrophic phenotype since 3-5 days post fertilization (dpf). Furthermore, we forced δ-KO zebrafish mutants to manifest a dystrophic phenotype by maintaining the larvae (immediately after hatching) in 1% Methylcellulose. Fish swimming in a viscous water experiment a stressful condition that exacerbates the skeletal muscle defects typical of sarcoglycanopathies. Evidence from the literature highlights the possible involvement of mitochondria in the pathogenesis of different types of muscular dystrophies, including sarcoglycanopathy, although it is still uncertain whether mitochondrial dysfunction occurs prior to myofiber necrosis or because of the onset of severe muscle damage. Currently, many small molecules that target mitochondria are being tested as potential treatments for muscular dystrophies. Thus, we decided to treat our novel sarcoglycanopathy models with one of such compounds. Diseased and control zebrafish embryos were treated following a weekly regimen with different concentrations of either the compound or vehicle. The effects of the small molecule on the skeletal muscle were evaluated by phalloidin staining and birefringence assay.