Abstract
Purpose :
The Leber’s congenital amaurosis (LCA) is one of the most severe genetic retinal dystrophies. The compound heterozygous mutations in the β subunit of chaperonin containing TCP-1 (CCT), encoded by the gene CCT2, were identified as LCA causative. The patients carrying these mutations exhibit a hypoplastic retina with typical LCA manifestations. The physiological role of mutated CCT2 in the retina remain unclear. To investigate possible functions of CCT2 that may be associated with the onset of LCA, we established a zebrafish model with cct2 mutation.
Methods :
A zebrafish line with cct2 mutation was established by CRISPR-Cas9. The phenotype was analyzed by microscopy, immunostaining, TUNEL and EdU/PH3 double labeling assays, and Western blotting at distinct larval stages. Phenotypic rescue was conducted by injection of CCT2 RNA. Chi-square test was used to validate the Mendelian inheritance and two-tailed Student's t-test was used for statistical analysis.
Results :
The zebrafish carrying the mutation in cctβ protein with the L394H replacement and deletion of 7 amino acid residues from 395- 401 (L394H-7del) was chosen to be analyzed. The homozygous of L394H-7del mutation exhibited a small eye phenotype at 2 days post-fertilization (dpf). The frequency of this phenotype was not significantly deviated from the Mendelian inheritance as judged from 5 independent mating (total 970 embryos; average incidence 25.3%; p-value of Chi-Square test 0.74). Injection of RNA encoding wild-type human CCTβ rescued the small eye phenotype and also rescued RGC
development and dramatically reduced cell death at 3 dpf while injection of RNA encoding LCA-causing mutant (T400P) or mock injections failed to rescue the phenotype. The differentiation of retinal ganglion cells (RGC) was attenuated, cell cycle was arrested, and the cell death occurred in the neural retina of the homozygous mutant at 2 dpf. The retinal structure was grossly disturbed, and it was difficult to distinguish the photoreceptor layer after 3 dpf. These results indicate that cctβ plays essential role in retinal cell cycle and RGC development.
Conclusions :
Dysfunction of cctβ leads to the hypoplastic retina that resembles retinal pathology of human CCT2-LCA patients. Injection of CCT2 mRNA dramatically rescued the hypoplastic retina. It is necessary to establish the mammalian model of CCT2 to validate the efficacy of gene therapy in future.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.