Abstract
Purpose :
Retinitis pigmentosa (RP), a common inherited retinal degeneration characterized by primary rod photoreceptor death, has been associated with mutations in the PDE6G gene, leading to autosomal recessive RP. However, the precise mechanism through which these mutations result in the disease phenotype remains elusive. This study introduces a zebrafish model with a knockout of pde6gb, a human PDE6G homolog, to investigate rod photoreceptor degeneration and utilizes the model to screen potential treatments promoting rod cell survival.
Methods :
A zebrafish pde6gb mutant was generated using CRISPR/Cas9 mutagenesis. Mutant founders were crossed with a transgenic line, Tg(rho:YFP-MTR), allowing for YFP labeling of rod photoreceptors, facilitating visualization and quantification of . Time-series automated reporter-based quantification in vivo (ARQiv) assays and confocal imaging were performed from 4 to 8 days post-fertilization (dpf) to assess rod cell loss. Quantitative PCR (qPCR) was performed to examine gene expressions in rod and cone photoreceptors. Immunofluorescence (IF) was conduct to analyze the cellular changes of mutant retinas at 8 dpf using 1d1 (rod), zpr1 (cone) and PCNA (proliferating cells) antibodies. Compounds including cell death inhibitors and reported neuroprotectants from other rodent models were evaluated in pde6gb mutant.
Results :
One frameshift allele pde6gbJH605A bearing a 10 bp deletion was identified. Homozygous mutant fish showed a progressive reduction in rod-YFP levels from 4 to 8 dpf compared to wildtype (WT), as confirmed by confocal imaging. qPCR demonstrated decreased expression of rod-specific genes, while cone marker genes showed no significant change. IF studies indicated rod cell loss without cone cell loss at the early developmental stage. Neuroprotective molecules, including a parthanatos inhibitor and three compounds known to promote rod cell survival in rodent models, were identified.
Conclusions :
This study successfully established a novel zebrafish pde6gb mutant, exhibiting progressive rod photoreceptor degeneration, mimicking a key pathological feature of RP. The model offers an excellent tool to explore the molecular mechanism of PDE6G-linked RP and serves as a valuable platform for drug discovery targeting RP.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.