Mechanism of cone and ganglion cell death

Title Mechanism of cone and ganglion cell death
Acronym HURET
Start date
End date
Sponsor European Research Council - Advanced Grant (ERC-AdG)
Institution Institute of Molecular and Clinical Ophthalmology Basel (IOB)

Associated cell lines

Project Description

1. Mechanism of starvation-induced cone death. The starting point is human retinal organoids in which cones are labeled with GFP. In preliminary experiments, we exposed the organoids to zero glucose medium and time-lapse imaged them in 3D using spinning disc confocal microscopy. The cones started to die after a one-day starvation, as evidenced by a decrease in the number of GFP-labeled cones. Using this paradigm, we will first isolate the surviving cones at 50% cone loss using fluorescence activated cell sorting and then perform single-cell RNA sequencing. Analysis of covariation of expressed genes will inform us about pathways involved in starvation-mediated cone death. A limitation of RNA sequencing is that it only indirectly reveals pathways via changes in gene expression and does not suggest molecules to interfere with the pathway. Therefore, we will perform a small-molecule screen using a library of 500 drug molecules for which the targets are known and that were selected to cover most known molecular targets. To do this, we have developed a method for making hundreds of organoids from the same iPSC line and organizing the organoids in multiwell plates. Each organoid will express GFP in cones. Using a 3D spinning disk microscope equipped with a motorized plate mover, we will image all organoids on five 96-well plate at fixed time points 12 hours apart, resulting in ~1.5 min imaging time per organoid. The effects of each molecule on organoids will be observed at three concentrations one log unit apart, with a few control organoids without drugs. These three batches of organoid plates will be imaged one after the other. We have developed software that can segment cone cell bodies and we will assess whether any of the drugs slow down cone degeneration in a dose-dependent manner. 2. Mechanism of pressure-induced ganglion cell death. High pressure in the eye is a risk factor in the development of glaucoma, where ganglion cells degenerate. After exposure to high pressure, we will perform a small-molecule screen in eye-brain organoids in which ganglion cells and their axons are present. We will create eye-brain organoids in which ganglion cells are labeled with GFP. To do this, we will screen our AAV promoter library for the labeling of ganglion cells and will also produce organoids in which we knock-in GFP after ganglion cell-specific genes using the 2A self-cleaving construct. We will then determine gene expression before ganglion cell death and screen 500 different drug molecules.