Use of Human Stem Cell-derived RGCs to Study the Mechanism of Optineurin-associated Glaucoma

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Project Summary/Abstract Glaucoma, which causes damage to and irreversible loss of retinal ganglion cells (RGCs), is a leading cause of blindness worldwide. In America, more than 3 million people are living with this disease. One form of glaucoma, normal tension glaucoma (NTG), in which there is loss of RGCs without evidence of increased eye pressure, is commonly associated with the optineurin (OPTN) E50K mutation. Studies in mice show that the E50K mutation can cause RGC death and optic nerve excavation. To date, all glaucoma therapy is directed at lowering eye pressure, not directly promoting RGC health and survival (neuroprotection). Here we have developed a method to differentiate and purify large amounts of RGCs from human embryonic stem cells to investigate molecular mechanism of optineurin-associated RGC injury. Optineurin is a critical player for mitochondrial degradation in the autophagy pathway, which is known as mitophagy. Our central hypothesis is that the stem cell derived RGCs with the OPTN E50K mutation will recapitulate RGC degeneration in glaucoma by disrupting the mitochondrial quality control (MQC) pathway. The proposed study is broken into three specific aims: 1) Determine the effect of OPTN E50K mutation on mitochondrial function and degradation in RGCs. 2) Investigate molecular mechanism of E50K mediated mitochondrial defect and perform a small molecule screen to find RGC protective compounds. 3) Model RGC degeneration in 3D retina with E50K mutation to study the effect of neuroprotective reagents. This proposal is innovative in multiple ways: first, unlike rodent models this stem cell derived RGC model is more likely to reflect human RGC biology. Second, we will investigate the molecular mechanism of MQC defect in glaucoma associated OPTN mutant in human RGC, which is recently indicated to be important for RGC biology and pathology. Third, a small molecule screen with a mutant reporter line will provide drug-screening platform for glaucoma as well as for other retinal diseases. This project is on track with the successful development of the RGC differentiation and purification method, flow based mitophagy assay and 3D retinal cup formation. We obtained OPTN mutants from our collaborator for the Aim1 and CRISPR based generation of OPTN mutant is underway for aim 2 and 3. PI's co- mentor Dr. Debasish Sinha is a leading scientist in autophagy field, whose office is in the same building and will provide all the support required during the mentored phase. Proposed project is deigned based on PI's expertise in cell biology, molecular biology, microscopy and stem cell along with ongoing training in retinal biology in Don Zack's lab (mentor). PI is in the ideal environment for the proposed research as Don Zack has an established stem cell based retinal research program along with state-of-the-art HCS facilities, stem cell core, several confocal and electron microscopes with renowned vision scientists available at Wilmer Eye/Hopkins. This will help PI to set-up collaboration and learn new techniques to built independent research program on mechanistic investigation of human retinal disease using stem cell derived disease models.