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Arupratan Das, Valentin Sluch, Chung-ha O. Davis, Nicholas Marsh-Armstrong, Donald J Zack; Use of Human Stem Cell-derived RGCs to Study the Mechanism of Optineurin-associated Glaucoma. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2536. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is a neurodegenerative disease of retinal ganglion cells (RGCs) leading to vision loss and, in some patients, ultimately to blindness. Optineurin (OPTN) mutants have been associated with normal tension glaucoma (NTG) in which there is RGC damage and cell loss without evidence of increased eye pressure. The E50K mutant allele of OPTN is associated with severe disease progression with reduction of retinal thickness and excavation of the optic nerve head. Moreover, the E50K mutation is associated with defects in mitochondrial quality control (MQC), which have been implicated in the pathogenesis of optic nerve degeneration. This study is based on the central hypothesis that OPTN-related glaucoma is due, at least in part, to a defect in MQC.
We have developed methods to differentiate and purify large amounts of human RGCs from pluripotent embryonic stem cells (hESCs). Purified RGCs derived from wild-type (WT), OPTN-/- and OPTN-E50K mutant hESCs are being used to study MQC by studying mitochondrial turnover (mitophagy) by flow-cytometry and mitochondrial metabolism by Seahorse technology (Agilent). Cellular defects in autophagosome formation around mitochondria in the E50K mutant are under investigation using high-resolution microscopy. RGC viability will be tested in the WT, OPTN-/- and E50K mutant cells with the goal of enhancing cellular life-span by rescuing the identified mitophagy defect in the OPTN mutants.
To test above hypothesis we have developed flow based mitophagy assay and observed 50% reduction in mitochondrial amount in presence of the mitophagy inducer drug (5mM CCCP) and further observed significant reduction in mitochondrial metabolism at different doses of CCCP. Using CRISPR/Cas9 based method we have created OPTN-/- reporter hESC line, which is verified by PCR, and sequencing.
Using CRISPR based mutagenesis technique we are further developing OPTN-E50K mutant hESC reporter line to assess MQC in RGC. LC3B lipidation is a bona fide marker for autophagosome formation; hence selective mitochondrial autophagy (mitophagy) defects are under investigation by LC3B lipidation status and localization of autophagy markers such as PARKIN, PINK, P62 and OPTN around mitochondria in the OPTN mutants. Such detailed molecular understanding will help identify novel therapeutic targets for the OPTN-related glaucoma.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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