Generating zebrafish model for studying the mechanism underlying the phenotypes of a rare genetic disease: N-glycanase deficiency
2Azrieli Faculty of Medicine, Bar Ilan University, Safed
Aim and Background: N-glycanase deficiency is a rare genetic disorder with fewer than 100 patients reported worldwide, and possibly many more undiagnosed persons suffering from this devastating disease with no effective treatment. The disease was linked to NGLY1, an enigmatic enzyme with multiple functions across a wide range of organisms. It catalyzes the de-glycosylation of misfolded glycoproteins, mediates signaling pathways, and is involved in regulation of mitochondrial physiology. We generated a novel zebrafish model for NGLY1 deficiency, to thoroughly study the pathophysiologic mechanisms involved in this rare disorder.
Methods: Zebrafish embryos were injected with Cas9 protein mix; DNA was extracted from injected embryos at 24h to determine mutagenesis efficiency. We crossed resulting F0 to WT and after series of crossings got homozygotes for a frame-shift mutation, ngly1(−/−). Nervous system morphology phenotypes were characterized using transgenic zebrafish line type Tg(HUC:gal4-uas:mem-YFP-mito:CFP). Muscle structure phenotypes were analyzed by Phalloidin staining, and skeletal traits by microCT. For studying the behavioral manifestations, we performed Danio-Vision (Noldus) motor test.
Results & Conclusions: We generated a viable zebrafish model for NGLY1 deficiency. The mutant fish manifests a significant loss of peripheral axons and muscle actin fibers, paired with movement elevation during stress condition. MicroCT of adult ngly1(−/−) fish showed craniofacial bones dislocation. The zebrafish model might facilitate comprehensive understanding of NGLY1 role in the development of the nerves and muscles. Studying both the juvenile and adult fish might shed light on the natural history of the disease and be used for the development of novel therapies.