Drug screening in a zebrafish model of Duchenne muscular dystrophy

Abstract

Two known zebrafish dystrophin mutants, sapje and sapje-like (sap c/100 ), represent excellent small-animal models of human muscular dystrophy. Using these dystrophin-null zebrafish, we have screened the Prestwick chemical library for small molecules that modulate the muscle phenotype in these fish. With a quick and easy birefringence assay, we have identified seven small molecules that influence muscle pathology in dystrophin-null zebrafish without restoration of dystrophin expression. Three of seven candidate chemicals restored normal birefringence and increased survival of dystrophin-null fish. One chemical, aminophylline, which is known to be a nonselective phosphodiesterase (PDE) inhibitor, had the greatest ability to restore normal muscle structure and up-regulate the cAMP-dependent PKA pathway in treated dystrophin-deficient fish. Moreover, other PDE inhibitors also reduced the percentage of affected sapje fish. The identification of compounds, especially PDE inhibitors, that moderate the muscle phenotype in these dystrophin-null zebrafish validates the screening protocol described here and may lead to candidate molecules to be used as therapeutic interventions in human muscular dystrophy.phosphodiesterase inhibitor | chemical treatment M uscular dystrophy is a disease in which the muscle forms normally at first but then degenerates faster than it can be repaired. The most common form of muscular dystrophy is Duchenne muscular dystrophy (DMD), representing more than 90% of the diagnosed cases. Mutations in the dystrophin gene were found to be the cause of both DMD and Becker muscular dystrophy (1, 2). Currently, prednisone is the only treatment option available for muscular dystrophy patients in the United States, although there are currently other options through approved clinical trials. Other treatments currently being tested or considered for treating muscular dystrophy include the small molecule PTC124, which promotes read-through of nonsense mutations (3), encouraging muscle development by myostatin down-regulation (4, 5), and the use of oligonucleotides to promote exon skipping to restore dystrophin expression (6).Recently, a number of chemical and drug screens have been published using zebrafish embryos (7-11). It is possible to quickly produce large numbers of mutant offspring that can then be assayed in multiwell plates and treated with different chemicals to determine if disease progression is modulated. Many of these screens have been highly successful in disease modeling (7) and drug screening (8-10), making the zebrafish ideal for highthroughput whole-organism screening of candidate compounds. Chemical compounds of relatively small molecular weight can bind to specific proteins and alter their function, resulting in nonheritable phenotype changes.In addition to their suitability for chemical screens, zebrafish also represent a good model to investigate genes involved in muscle development and degeneration, including human muscular dystrophy (12-18). The orthologs of many dystrophin-glycoprot...

MicroRNA-199a is induced in dystrophic muscle and affects WNT signaling, cell proliferation, and myogenic differentiation

“…The sapje dystrophinmutant zebrafish model is an excellent tool for studying dystrophic-muscle and disease progression, as the sapje mutants show dystrophic disease severity and can be analyzed in large numbers in a short period of time. 16,17 The sapje dystrophic zebrafish has a more severe muscle phenotype in which their dorsal muscles progressively waste resulting in significant early lethality as compared with mdx mouse models. [16][17][18] The sapje zebrafish have weakened and degenerating muscle and the majority of mutants (95%) expire by 10 dpf due to the inability to swim and oxygenate their muscles.…”

“…16,17 The sapje dystrophic zebrafish has a more severe muscle phenotype in which their dorsal muscles progressively waste resulting in significant early lethality as compared with mdx mouse models. [16][17][18] The sapje zebrafish have weakened and degenerating muscle and the majority of mutants (95%) expire by 10 dpf due to the inability to swim and oxygenate their muscles. Thus, we chose to utilize the sapje dystrophic zebrafish as a model for analyzing dysregulated dystrophic miRNAs and to expand our previous findings obtained from DMD muscle biopsies.…”

Research and Development of Biotechnologies Using Zebrafish and Its Application on Drug Discovery

“…These results indicate that the protein O-mannosyltransferase machinery in zebrafish and humans is conserved and suggest that zebrafish may be useful for functional studies of protein O-mannosylation. More recently, Dr. Kunkel's group has reported that two known zebrafish dystrophin mutants, sapje and sapje-like (sapc/100), represent excellent smallanimal models of human muscular dystrophy 51 . Using these dystrophin-null zebrafish, they have screened the Prestwick chemical library for small molecules that modulate the muscle phenotype in these fish.…”

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

“…Due to the fact that most muscular dystrophies result from mutations in proteins that participate in the link between the extracellular matrix and the sarcolemmal cytoskeleton, a common observation at the cellular level is the loss of sacrolemmal integrity 8,9 . This understanding of the primary pathomechanism(s) associated with muscular dystrophies is the product of numerous years of research employing animal model systems 2,[10][11][12][13][14][15] . However, despite advances in the field, there are still limited therapeutic options for treatment or management of the range of dystrophy subtypes.…”

“…To date, zebrafish have proven a valuable tool in disease research. The zebrafish model has been used to identify and validate novel human disease causing mutations 16,17 , elucidate uncharacterized disease causing mechanisms 17,18 , and identify novel therapeutic strategies 12,19 . These advances were made, in part, by the canonical strengths of the zebrafish system such as their optical clarity, ease of genetic manipulation, and ability to breed in large numbers 20 .…”