Abstract
Purpose :
Optic atrophy (OA) is a form of inherited optic neuropathy, characterised by progressive and irreversible degeneration of retinal ganglion cells (RGCs). This degeneration subsequently leads to sight loss, for which there is presently no treatment. Most OA patients have mutations in the mitochondrial fusion protein OPA1. It is unclear why OPA1 mutations lead to RGC death and subsequent vision loss.
Methods :
CRISPR/Cas9 gene editing was used to disrupt the endogenous Opa1 gene of Fruit flies (Drosophila melanogaster) and zebrafish (Danio rerio). The fly knockout (KO) model was generated using the UAS-GAL4 system to create neuron-specific expression of CRISPR guides. In zebrafish, CRISPR guides injected into embryos created an 800 bp deletion in the Opa1 GTPase domain. PCR and qPCR validated editing events in both models. In flies, fluorescence microscopy was used to assess mitochondrial morphology. Optokinetic response assays were used to assess vision in zebrafish. Transmission electron microscopy and Seahorse extracellular flux assays were used to examine mitochondrial morphology and function in zebrafish larvae. Fluorescence microscopy was used in conjunction with the Tg(isl2b:mitoeGFP-2ATagRFPCAAX) line to look at RGC morphology in KO zebrafish larvae. Mass spectrometry was used for proteomic analysis of both models.
Results :
qPCR confirmed reductions in Opa1 expression in both the Crispant (~75%, p<0.01) and fruit fly (~35%, p<0.01) models. Neuron-specific KO flies have more rounded mitochondria (p<0.0001). Zebrafish opa1 crispants display a significant (p<0.01) loss of visual acuity compared to buffer-injected siblings. Crispant larvae have dysfunctional respiration according to several metrics, including a 15% reduction in maximal respiration (p<0.05). Provisional TEM results suggest accumulation of rounded mitochondria in the optic nerve of zebrafish larvae, which was corroborated by fluorescence imaging. Proteomic analysis indicated mitochondrial dysfunction in the crispants, which was confirmed through seahorse assays, that showed various respiratory defects including reduced basal respiration (p<0.01).
Conclusions :
Opa1 loss of function models were created in both zebrafish and flies. These will be used to study the pathological mechanisms underlying Opa1-mediated vision loss. The phenotypic changes described can be used for screening potential therapies for Opa1-linked OA.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.