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Abstract #127366 Published in IGR 25-3
Methods for Mitochondrial DNA Damage and Depletion in Immortalized Trabecular Meshwork Cells
Kennedy SP; Tsaturian E; Zhao L; Morgan JTInternational journal of molecular sciences 2025; 26:
Mitochondrial DNA (mtDNA) damage in trabecular meshwork (TM) cells occurs in open-angle glaucoma (OAG). However, current in vitro models for OAG-like changes in TM cells do not explicitly incorporate mtDNA damage. This work validated two methods of mtDNA damage in immortalized TM cells and assessed OAG-associated expression changes. mtDNA was depleted in TM-1 cells via both ethidium bromide (EtBr) treatment and doxycycline (Dox) induction of a mutant (Y147A) version of Uracil DNA Glycosylase 1 (UNG1) in TM-1 cells (TM-1). Levels of mitochondrial proteins (ATP5F1A, COXII, and COXIV) were measured via western blot. mtDNA levels and mRNA for OAG-associated transcripts (, , , and ) were measured by qPCR. There was a statistically significant decrease in mtDNA levels per cell at all treatment times in both EtBr-treated TM-1 cells and induced TM-1 cells. Protein levels of ATP5F1A were not significantly changed; COXII and COXIV showed significant decreases after both EtBr and Dox induction. Both models resulted in upregulation of , , and ; additionally, EtBr treatment but not Dox induction resulted in upregulation. In conclusion, two models of mitochondrial depletion were demonstrated in immortalized TM cells; damage was associated with increases in OAG-associated transcripts, supporting a link between mitochondrial damage and glaucoma phenotypes.
Department of Cell, Molecular, and Developmental Biology, University of California, Riverside, CA 92521, USA.
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