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Henry Tseng, Emily Sun, James Powers, Charlaine Chen; Loss of optineurin disrupts mitochondrial networks and morphology. Invest. Ophthalmol. Vis. Sci. 2019;60(9):663.
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Mutations in the autophagy-gene optineurin (OPTN) are associated with primary open-angle glaucoma and amyotrophic lateral sclerosis, but the pathophysiological mechanism is unclear. Recent studies have demonstrated that OPTN may play an important role in regulating mitochondrial homeostasis. Our research tests the hypothesis that loss of normal OPTN function disrupts mitochondrial networks and morphology.
We used primary mouse embryonic fibroblasts (MEFs) purified from wild-type (WT) OPTN, E50K OPTN and OPTN knock-out (KO) transgenic mice previously generated in our laboratory. The cells were treated with oligomycin and antimycin (OA), which are inhibitors of mitochondrial respiration, at varying time lengths. Immunofluorescent staining of mitochondria using MitoTracker and antibodies to TOM20 followed by confocal microscopy was used to visualize morphology. Quantification of mitochondrial morphology was performed using ImageJ and Mitochondrial Network Analysis software.
At baseline, E50K OPTN MEFs had a larger average cell size than WT and KO OPTN cells. When normalized to average cell size, WT OPTN MEFs exhibit a complex mitochondrial network with fewer individual mitochondrial puncta than E50K (p<0.05) and KO OPTN cells (p<0.01). As such, compared to E50K and KO OPTN cells, WT OPTN mitochondrial networks had more branches (p< 0.05 and p<0.01, respectively) and greater branch lengths (p<0.01 and p<0.00001). OA treatment resulted in mitochondrial fragmentation, with decreased number of mitochondrial networks and increased number of punctate mitochondria in all three cell groups.
Our data showed that WT OPTN cells had larger and more branched mitochondrial networks, while E50K and KO OPTN cells contained mitochondria that are more fragmented. This supports the hypothesis that OPTN plays a role in maintaining a healthy mitochondrial network. Future work will be needed to determine if fragmentation and dysfunction of mitochondrial network might increase oxidative stress and cellular apoptosis as a possible mechanism for retinal ganglion cell neurodegeneration in glaucoma.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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