Abstract
Purpose :
Optic atrophy (OA) is the most common form of inherited optic neuropathy. OA is characterised by progressive and irreversible degeneration of retinal ganglion cells (RGCs). Loss of RGCs is associated with a gradual loss of vision, usually beginning insidiously in childhood. Approximately 60-70% of OA patients carry mutations in the mitochondrial fusion protein OPA1. However, it is unclear why OPA1 mutations lead to RGC death and subsequent vision loss.
Methods :
Fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio) models of OA were developed using CRISPR/Cas9 gene editing to create functional knockouts (KO) of endogenous Opa1. The fly model was generated using the UAS-GAL4 system to create neuron-specific expression of CRISPR guides. In zebrafish, CRISPR guides were injected into zebrafish embryos to create an 800 bp deletion in the Opa1 GTPase domain. PCR and qPCR validated Opa1 editing events in both models. Sanger sequencing verified the deletion in zebrafish. In flies, survival was monitored compared to control animals. In 5-day old zebrafish, visual behaviour was measured using optokinetic response (OKR) assays and retinal morphology was examined by light microscopy.
Results :
Genomic deletion events were present in 93.26% of sampled zebrafish larvae (n=260). DNA sequencing confirmed the deletion creates an early truncation in zebrafish Opa1. qPCR revealed a ~75% reduction in zebrafish Opa1 mRNA (p<0.01) in animals with confirmed deletion events relative to siblings injected with Cas9 working buffer. In neuron-specific KO flies, a ~35% reduction of Opa1 transcript was identified in fly heads (p<0.01). Neuron-specific KO flies demonstrate a significantly reduced median life expectancy of 2 days post-pupation (p<0.0001). Zebrafish opa1 crispants display a significant (p<0.01) loss of visual acuity compared to buffer-injected siblings. Preliminary data indicates a reduction in thickness of the inner and outer plexiform layers of the retina.
Conclusions :
Opa1 loss of function models were created in zebrafish and flies. These in vivo models will be used to study how Opa1 related mitochondrial dysfunction can lead to vision loss. Phenotypic changes linked to loss of Opa1 can be used for screening potential therapies for Opa1-linked OA. In future, proteomics will be implemented to examine changes induced by Opa1 deficiency in both models.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.