A limb-girdle muscular dystrophy 2I model of muscular dystrophy identifies corrective drug compounds for dystroglycanopathies

Abstract

Zebrafish are a powerful tool for studying muscle function owing to their high numbers of offspring, low maintenance costs, evolutionarily conserved muscle functions, and the ability to rapidly take up small molecular compounds during early larval stages. Fukutin-related protein (FKRP) is a putative protein glycosyltransferase that functions in the Golgi apparatus to modify sugar chain molecules of newly translated proteins. Patients with mutations in the FKRP gene can have a wide spectrum of clinical symptoms with varying muscle, eye, and brain pathologies depending on the location of the mutation in the FKRP protein. Patients with a common L276I FKRP mutation have mild adult-onset muscle degeneration known as limb-girdle muscular dystrophy 2I (LGMD2I), whereas patients with more C-terminal pathogenic mutations develop the severe Walker-Warburg syndrome (WWS)/muscle-eye-brain (MEB) disease. We generated fkrp-mutant zebrafish that phenocopy WWS/MEB pathologies including severe muscle breakdowns, head malformations, and early lethality. We have also generated a milder LGMD2I-model zebrafish via overexpression of a heat shock-inducible human FKRP (L276I) transgene that shows milder muscle pathology. Screening of an FDA-approved drug compound library in the LGMD2I zebrafish revealed a strong propensity towards steroids, antibacterials, and calcium regulators in ameliorating FKRP-dependent pathologies. Together, these studies demonstrate the utility of the zebrafish to both study human-specific FKRP mutations and perform compound library screenings for corrective drug compounds to treat muscular dystrophies.

Discovery of Novel Therapeutics for Muscular Dystrophies using Zebrafish Phenotypic Screens

“…Zebrafish co-injected with human FKRP p.Leu276Ile mutation mRNA and an anti-sense morpholino oligonucleotide targeting the endogenous zebrafish fkrp gene showed a mild dystrophic phenotype, consistent with the human disorder [109]. Based on these findings, a zebrafish model of LGMD2I was developed using heat-shock inducible expression of the human p.Leu276Ile mutation in an [120]. Chemicals were screened at 2 dpf, 1 day after heat shock-inducible over-expression of a human FKRP transgene.…”

“…To address whether the therapeutic candidates identified in the short-term screen could reverse pathology once it had become established, a longterm screen was conducted in which 4-5 dpf Note that the composition and number of chemicals in a library can change over time. LGMD2I [99,120]. See text for details.…”

“…Abbreviations: PDE, phosphodiesterase inhibitor. FKRP-deficient zebrafish background [120]. This fish was characterized by abnormal birefringence indicative of myofibrillar disorganization, impaired spontaneous and touch-evoked swimming performance, reduced axial muscle tetanic force, eye and brain malformations, pericardiac edema, and a reduced lifespan.…”

“…In order to identify potential new therapeutics, a two-stage screening approach, similar to the method previously used on sapje and sapje-like larvae [77,121], was used to identify small molecules that reduced the pathological phenotypes of the LGMD2I zebrafish model [120]. The first stage of the screen identified 20 unique compounds from the Prestwick 2 collection that reduced the occurrence of abnormal birefringence, eye and brain abnormalities, pericardiac edema, and depressed vitality from an expected 50% of larvae to <10% (Table 3).…”

Zebrafish as a Model for the Study of Lipid-Lowering Drug-Induced Myopathies

“…The rapid screen of bioactive compounds, able to modulate pathological phenotype and allowing identification of novel drugs or dietary supplements, is one example. Such screens were shown to be successful in drug re-targeting, identifying the FDA-approved drugs as a candidate to e.g., treat Duchenne [ 161 , 162 ] and limb girdle muscular dystrophies [ 163 ]. The drug re-targeting approach allows for time and cost-saving, but also opens the possibilities of development of new therapeutic strategies [ 147 ].…”