The retinal pigment epithelium (RPE) is often the initial site of pathogenesis in many retinal degenerative diseases. The proposed study aims to understand how mitochondrial dynamics responds to and affects RPE function and health. The successful completion of the project will provide additional insights into disease mechanisms. The RPE performs many important functions to ensure photoreceptor homeostasis, including daily phagocytosis of photoreceptor outer segment. Phagocytosis is a metabolically challenging process. The formation, transport and degradation of large numbers of phagosomes are energy intensive for the RPE, as each RPE cell interacts with up to 200 photoreceptor cells. On the other hand, the products of phagosome degradation could represent an energy source. We therefore speculate that, RPE mitochondria have to respond and adapt to different daily metabolic events. In many degeneration diseases, impaired mitochondrial functions have been observed, and this is often associated with defects in mitochondrial dynamics. However, little is known about mitochondrial dynamics in RPE. In this study, we propose to use super-resolution high-speed live imaging and electron microscopy to study the dynamics of RPE mitochondria, including morphology (fission/fusion), distribution and motility. To complement these studies, we will also conduct metabolic analysis such as Seahorse assay and Fluorescent Lifetime Microcopy to measure mitochondrial functions. We will focus on answering three questions: 1) How does RPE phagocytosis affect mitochondrial dynamics? 2) What regulatory machineries drive mitochondrial dynamics? 3) How are RPE mitochondria dynamics affected in the inherited retinal degeneration, choroideremia? To ensure relevance to human health and disease, we will compare and contrast our observations in mice with those in human RPE cell lines, as well as patient-derived iPSC-RPE cultures. By imaging with very high spatial and temporal resolutions, the study will provide novel insight into RPE mitochondrial dynamics, which in turn will allow us to uncover new disease mechanism and identify novel therapeutic targets. The scope and potential public health impact of the proposed study fits well with the missions of NEI to help prevent and treat eye diseases. The proposed project also provides invaluable training opportunity for the applicant towards her long- term career goal to become an independent investigator in molecular and cellular vision research. The applicant will gain knowledge of the vision system, specifically the interaction between photoreceptors and RPE. The applicant will also receive training on various cutting-edge microscopy techniques, as well as patient-derived iPSC as a model to study disease mechanism in vitro. Together with her prior experience in vascular biology, the training proposed here will set the applicant apart in a uniquely qualified position to study molecular mechanisms and cell-cell interactions involved in health and diseases of the vision system.
Recent work has emphasized the importance of RPE metabolism in retinal function and health. However, little is known about the cell biology of RPE mitochondria. The proposed study aims to employ state-of-the-art methods, including high-speed live- cell imaging to investigate how RPE mitochondrial dynamics responds to different conditions.
