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

## TL;DR

This study introduces two methods to damage mitochondrial DNA in lab-grown eye cells, linking mitochondrial damage to glaucoma-related changes.

## Contribution

The paper introduces two validated methods for inducing mitochondrial DNA damage in immortalized trabecular meshwork cells.

## Key findings

- Both ethidium bromide and doxycycline-induced UNG1Y147A significantly reduced mtDNA levels in TM cells.
- Mitochondrial protein COXII and COXIV levels decreased significantly in both models.
- OAG-associated genes CTGF, FN1, and PAI1 were upregulated in both models, with SFRP1 upregulated only in ethidium bromide-treated cells.

## Abstract

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-1rtTAadv-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-1rtTAadv-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.