Description
Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in adults over 60 years. It can be classified into two main forms: neovascular and dry AMD. The hallmarks of dry AMD are the accumulation of drusen (yellow deposits composed of lipids and protein) and the disruption of the retinal pigmented epithelium (RPE) cells and photoreceptors, leading to cell death. The late-stage, geographic atrophy, is characterised by progressive, expanding lesions in the retina that critically affect the macula, resulting in central vision loss.
Mitochondrial dysfunction in RPE cells is increasingly implicated in retinal degeneration; however, the cellular pathways that determine RPE survival or death under mitochondrial stress remain poorly understood. Interestingly, around 20% of patients with maternally inherited diabetes and deafness (MIDD), caused by the m.3243A>G mitochondrial DNA mutation, develop a geographic atrophy like phenotype. Reports have shown that patients mitochondrial tRNA Leucine (UUR) lacks the tm5U wobble uridine modification, performed by GTPBP3 and MTO1 enzymes.
The aim of this project is to develop a cellular model of mitochondrial dysfunction and use that for a whole-genome CRISPR knockout screen to systematically identify genetic modifiers of RPE vulnerability.
We have successfully knocked out GTPBP3 in ARPE-19 cells using CRISPR-Cas9 and validated at the DNA, RNA and protein level. Functionally, the OXPHOS complex subunits abundance was reduced, particularly of Complex I and Complex IV, in line with published studies. Seahorse XF Mito Stress test demonstrated significantly reduced basal/maximal respiration and ATP production in the knockout compared to the wild type. Finally, XTT metabolic test demonstrated that the knockout metabolic activity is significantly reduced in challenging conditions (galactose). In parallel, the GeCKO v2 whole-genome CRISPR library was successfully amplified and validated, meeting all quality control criteria for downstream screening. We have also successfully completed the pre-screen tests (seeding density, antibiotic resistance and multiplicity of infection) and are currently in screening phase.
Together, these results establish a scalable and phenotypically validated MIDD-like RPE model necessary to conduct an unbiased genome-wide screen for genetic modifiers of mitochondrial dysfunction, with the long-term goal of identifying novel therapeutic entry points for geographic atrophy.
Lay Abstract
Age-related macular degeneration (AMD) is one of the main causes of sight loss in people over 60. In the “dry” form of AMD, fat and protein deposits disrupt the cells at the back of the eye which gradually become damaged and die. In advanced stages, called geographic atrophy, this damage spreads across the retina making everyday activities progressively more difficult. Currently, there are limited treatments available.
Our research focuses on the role of mitochondria, the “energy factories” of cells, which have increasingly been linked to retinal diseases, including AMD. Interestingly, some patients with a rare inherited mitochondrial condition called maternally inherited diabetes and deafness (MIDD) develop retinal changes similar to geographic atrophy.
To better understand why retinal cells become vulnerable, we created a laboratory model using retinal pigment epithelial (RPE) cells with a disrupted gene called GTPBP3. These cells showed reduced energy production and poorer survival under stress, closely resembling mitochondrial dysfunction seen in patient cells.
We are now using a genome-wide CRISPR screening approach to identify genes that may protect or harm retinal cells under mitochondrial stress. In the future, this work could help uncover new treatment targets for geographic atrophy and other retinal degenerative diseases.
| Lay Title | Mitochondrial Dysfunction in Age-related macular degeneration |
|---|---|
| Role | PhD Student |