Abstract
Purpose :
Although disease risk variants and associated genes in age-related macular degeneration (AMD) have been identified by multiple studies, the key pathophysiological pathways underlying disease pathogenesis remain to be elucidated. This gap is mainly attributed to the lack of reliable animal models to dissect the functional impact of these risk genes. This study aims to elucidate the molecular mechanisms of AMD progression in a human context by establishing a novel aged retina model using pluripotent stem cells.
Methods :
Human pluripotent stem cells were differentiated into retinal organoids using our established protocol, with minor changes to increase the number of cones. Proteasome inhibitor lactacystin was applied to mature organoids to induce aging phenotypes. To determine whether our aged cone-enriched model is able to mimic AMD pathogenesis, we evaluated the impact of AMD risk gene RDH5, whose reduction in patients and animal models is associated with macular degeneration in elderly. As RDH5 catalyzes the last step to generate 11-cis retinal for photoreceptors, the isomer 9-cis retinal was withdrawn from the cultures for two weeks. The length of photoreceptor outer segments in aged cone-enriched retinal organoids were quantified. The organoids were then harvested for immunostaining, immunoblotting, and mass spectrometry. All experiments were performed in 3 independent batches, each of which had at least 5 organoids (n≧15).
Results :
In the absence of 9-cis retinal, lactacystin-treated aged photoreceptors displayed a significant reduction in outer segment length compared to the untreated young ones (88.62±32.50mm vs 140.95±30.84mm, p= 0.024), which mimic the phenotypes in early to intermediate AMD patients. When 9-cis retinal was supplemented, young and aged photoreceptors had comparable outer segment length (112.491±23.86mm vs 109.50±27.28mm, p= 0.844). Protein analyses revealed dysregulation of autophagy in aged cone-enriched retinal organoids in the absence of 9-cis retinal, which could contribute to AMD pathology.
Conclusions :
Aged cone-enriched retina displayed shorter outer segments in the absence of 9-cis retinal, mimicking phenotypes in AMD patients and suggesting a promising model to interrogate the role of risk genes in disease progression. Future studies will compare our model system with AMD patient retina and investigate the underlying mechanisms of risk genes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.