Abstract
Purpose :
Retinal degeneration involves different underlying causes; including environmental insults and genetic mutations. Photoreceptors (PR) and RPE require large amounts of ATP. But mitochondrial function and ATP production are sensitive to environmental challenges and aging; tissues from elderly patients show a decrease in mitochondrial health and ATP. Here we established a molecular mechanism for a novel compound identified in a high-throughput-screen designed to identify agents effective in slowing degeneration in a mitochondria-dependent manner.
Methods :
Mouse 661w cone and human ARPE-19 cells were used. Toxic challenges mimic conditions in retinitis pigmentosa (high Ca2+ and oxidative stress in PR) and AMD (serum starvation in RPE). Cell viability was measured using MTT assays; mitochondrial respiratory capacity via Seahorse assays; click chemistry was used to identify the target for the lead candidate MC16; relevant proteins were analyzed via Western blots (cell lysates or immunoprecipitation); and mitochondrial morphology and protein distribution was visualized by MTDR and immunohistochemistry. Structure and function of the rd10 retina upon MC16 treatment was analyzed by histology and electroretinography.
Results :
MC16 increased cell viability and OXPHOS in cells exposed to toxicants. Click chemistry identified the target for MC16 as FUS and its binding partner Hsp60. Confocal microscopy documented that in oxidatively stressed cells, FUS exited the nucleus and associated with or formed aggregates on mitochondria; treatment with MC16 prevented the nuclear egress of FUS, preserved mitochondrial fusion status and prevented the association of FUS with mitochondria. Immunoprecipitation revealed that FUS forms condensates on mitochondria based on its interaction with Hsp60. In vivo, treatment of the rd10 retina with MC16 eyedrops increased photoreceptor cell survival and cone function.
Conclusions :
Based on the identification of MC16 as a molecule that extends lifespan of cells in a mitochondria-dependent manner, the identification of FUS as its target and the knowledge of FUS as a transcription factor and a component in biomolecular condensates, we suggest that MC16 acts by contributing to expression of genes involved in anti-oxidant response and mitochondrial homeostasis, and by preventing condensate formation and mitochondrial dysfunction.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.