Methods for Mitochondrial DNA Damage and Depletion in Immortalized Trabecular Meshwork Cells

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^{rtTAadv-TRE-UNG1Y147A}). Levels of mitochondrial proteins (ATP5F1A, COXII, and COXIV) were measured via western blot. mtDNA levels and mRNA for OAG-associated transcripts (CTGF, FN1, PAI1, and SFRP1) 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^{rtTAadv-TRE-UNG1Y147A} 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 CTGF, FN1, and PAI1; additionally, EtBr treatment but not Dox induction resulted in SFRP1 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.