Author(s): Avery Zentner, Kasia Poulson, Syrus Miner
Mentor(s): Hung-Yu Shih, Yu-Chien Liu
Institution UTech
4H Leukodystrophy (4H) is an early-onset rare genetic disease, with no current cure. 4H patients exhibit a broad spectrum of hypomyelination, hypodontia, and hypogonadotropic hypogonadism. Mutations in subunits of the RNA polymerase III (POLR3) could lead to 4H. Polr3a and Polr3b are the largest and second-largest subunits of the POLR3 complexes with various mutations identified in 4H patients. Although there are several mouse models established, the phenotype development needs several weeks. This is not ideal for small molecule compound screening. Zebrafish, Danio rerio, could be a complemental model to the existing models to study the pathogenesis and develop the therapy of 4H. Zebrafish have emerged as the model for leukodystrophy. This project aims to establish and characterize the zebrafish 4H model by knocking out the largest subunit, RPC1, which is encoded by the polr3a gene, with the CRISPR/Cas9 approach. The polr3a Crispants showed a significant increase in mortality and a decrease in mobility. Gene expression analysis revealed a reduction in oligodendrocyte, myelin-associated, and pituitary gland-related genes. By contrast, the Alizarin Red staining showed no change in the number of teeth. TUNEL staining assay revealed a significant increase of apoptotic cells in the polr3a Crispants. Our current findings suggest the polr3a knockout of zebrafish could develop hypomyelination and hypogonadotropic hypogonadism of 4H through increasing cell apoptosis. Future studies aim to address the specific impacts of Polr3a deficiency on the oligodendrocytes, myelinated oligodendrocytes, and pituitary gland cells and the underlying mechanisms of 4H development and progression. Moreover, we will utilize our 4H zebrafish models for therapeutic drug screening.