Vulnerable and Resilient Cells in Retinal Degeneration

PROJECT SUMMARY Inherited retinal degeneration (IRD) is a group of genetic diseases featuring progressive loss of photoreceptor neurons in the retina and eventual blindness. Affected photoreceptors largely die through apoptosis that was once thought to be irreversible. However, recent studies in different systems have reported a phenomenon called “anastasis”, in which some cells that received an apoptotic signal become resilient and return to a healthy state. The presence of resilient photoreceptor cells after light damage was suggested by several studies, but there is no clear data on photoreceptors in retinal degeneration or knowledge of underlying cellular/molecular mechanisms. This application aims to close this knowledge gap by testing our hypothesis that each photoreceptor cell has an intrinsic capacity to “self-repair” or recover from apoptosis, and that therapeutic treatments enhancing this capacity will maintain photoreceptors in IRDs. To test our hypothesis, we will use mouse models to identify resilient vs. vulnerable rod/cone cells during retinal degeneration. We have established an in vivo genetic tool, the living-color reporter mouse line, named CaspBiosensors. In this model, we take advantage of a well-known apoptotic marker, activated Caspases 3/7 (Casp) to label cells that undergo apoptosis (present) with red fluorescence and cells that have survived apoptosis (past) with green fluorescence. Our preliminary results demonstrate that we can detect present/past Casp+ photoreceptors in a relevant model by both imaging in live animals and histological sections. In this grant, we propose experiments to confirm and expand these findings at the cellular and molecular levels. Specific Aim 1 will determine the survival outcome of vulnerable (red) and resilient cells (green) under environmental (light damage) and genetic (three IRD models) insults, using chronic in vivo imaging-based analyses complemented by histological examination at end-points. Specific Aim 2 will identify molecular signatures of cellular resiliency using single-cell RNA sequencing. We will validate selected candidate genes using loss-of-function and gain-of- function manipulations. We will also investigate the capacity/mechanisms of gene therapy and a proven neuroprotection reagent in preventing photoreceptors degeneration in IRD models. The outcome of this research is expected to significantly impact our understanding of apoptotic photoreceptor degeneration in IRDs and the development of more effective therapeutic approaches. Improving long-term efficacy of gene therapy is essential as photoreceptors have continued to degenerate even in the few successful ocular gene therapy clinical trials. Our study will inform potential improvements by adding neuroprotection strategies to prolong the life of targeted photoreceptors.