Modeling Sarcoglycanopathy in Zebrafish

Sarcoglycanopathies are rare autosomal recessive disorders belonging to the limb girdle muscular dystrophy family (LGMD). They are caused by mutations in sarcoglycan genes (SGCA, SGCB, SGCG, SGCD) coding for α- β- γ- δ- sarcoglycan (SG), respectively. SGs form a key structural complex of the plasma membrane of skeletal and cardiac muscle. Mutations in one SG gene result in the absence or strong reduction of the corresponding protein. This destabilizes the entire SG-complex with a consequent reduction of the membrane stability, which progressively leads to muscle degeneration. Nowadays there is no effective treatment for these rare diseases. Since sarcoglycanopathies are still without an effective treatment, any effort aimed at improving our understanding of their pathogenic mechanisms may help identify new potential therapeutic targets. To this intent, the availability of in vivo models capable of reproducing both the structural and functional consequences of sarcoglycan deficiency is essential. The overall goal of this PhD project was the generation and characterization of new zebrafish lines, models of β- and δ-SG deficiency, with the final aim of establishing a robust vertebrate platform for phenotype-based drug screening. Using CRISPR/Cas9 genome editing, two knockout (KO) lines (sgcb⁻/⁻ and sgcd⁻/⁻) and one knock-in (KI) line carrying the E264K missense mutation in sgcd (sgcdE264K/E264K) were successfully generated. The KO models emulate sarcoglycanopathy cases in which the absence of the SG protein results from large gene deletions or out of frame/null mutations. Conversely, the KI line is intended to mirror cases caused by missense mutations, which, incidentally, account for the majority of gene defects in sarcoglycanopathies. Despite the complete absence of β-SG and δ-SG in sgcb⁻/⁻ and sgcd⁻/⁻ and the severe reduction of δ-SG in sgcdE264K/E264K, larvae up to 5 days post fertilization (dpf) exhibited only a mild myopathic phenotype under standard conditions. This observation is consistent with the progressive nature of the disease, and in fact, both KO and KI zebrafish developed a clear dystrophic phenotype in adulthood. An advantage of using zebrafish in phenotype-based drug screening is the possibility to carry out experiments during the early stage of development. However, the lack of an evident phenotype, prompted us to develop a strategy to unmask initial muscle defects. Given that destabilization of the SG complex is expected to weaken the sarcolemma, we hypothesized that subjecting larvae to mechanical overload would induce muscle damage. To this end, we created a viscous environment (by adding 1% methylcellulose in the fish water) to increase swimming effort. This approach produced clear, quantifiable and reproducible stress-induced muscle damage and impaired swimming performance, making it suitable for high-throughput applications. Altogether, our findings show that the new zebrafish lines recapitulate key pathological features of sarcoglycanopathies across developmental stages, thus representing powerful tools for studying disease onset and progression. Furthermore, the development of an early stress-induced phenotype makes these models a valuable platform for testing therapeutic compounds and for large phenotype-based drug screening. As proof of concept, we successfully observed a recovery of the myopathic phenotype, by treating larvae with MG132, a well-known proteasome inhibitor previously shown to reduce muscle damage in different zebrafish models of muscular dystrophy.