Abstract
Purpose :
How retinal ganglion cells (RGC) degenerate in glaucoma remains unclear, especially without elevated intraocular pressure. To uncover a possible mechanism, we studied the optineurin (OPTN) gene whose genetic mutations (specifically an E50K mutation) have been found in familial normal tension glaucoma patients. Using cells from transgenic OPTN E50K and knock-out mice generated in our laboratory, we discovered abnormal mitochondrial morphologies. Due to the complex curvilinear tubular structure, mitochondrial networks have been difficult to analyze and compare quantitatively. In this work, we describe a novel method for mitochondrial analysis and found that mutant E50K OPTN and loss of OPTN led to increased mitochondrial fission and fragmentation.
Methods :
We developed a novel approach, called the Multiparameter Persistence Model, and software for quantitative analysis of fluorescently labeled mitochondria networks. To test this, we treated cells with oligomycin and antimycin A (OA), which blocks mitochondrial oxidative phosphorylation to simulate oxidative stress in the mitochondria. Treatment was applied to mouse embryonic fibroblasts (MEFs) purified from our OPTN transgenic mice with E50K OPTN and knock-out of OPTN and compared to control wild-type cells. Mitochondria was immunostained using a Tom20 antibody followed by confocal microscopy imaging and analysis.
Results :
Our novel approach for mitochondrial quantitation using the Multiparameter Persistence Model overcomes many limitations present in current analytical methods. Using this methodology, we found increased mitochondrial fragmentation and fewer network branches in MEFs from E50K or knock-out OPTN mice. These findings were not worsened by OA treatment. Our data suggest that abnormal OPTN function increases mitochondrial fission and disrupts mitochondrial networks.
Conclusions :
Our data strongly suggest that OPTN plays an important role in mitochondrial homeostasis. This illustrates a possible mechanism in which a genetic insult in OPTN disrupts mitochondria function, which in turn leads to RGC dysfunction and degeneration in glaucoma. Targeting this pathway via OPTN might pave the way for future advancements in the treatment of glaucomatous neurodegeneration.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.