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Overview of DNA Repair

In order to be passed through generations, genomic DNA must be undamaged and error-free. However, every day, DNA in a cell undergoes several thousand to a million damaging events by natural causes and external factors. Ionizing radiation such as UV rays, free radicals produced during cellular respiration, and hydrolytic damage from metabolic reactions can alter the structure of DNA. Damages caused include single-base alteration, base dimerization, chain breaks, and cross-linkage.
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Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue
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Methods for Mitochondrial DNA Damage and Depletion in Immortalized Trabecular Meshwork Cells.

Shane P Kennedy1, Emily Tsaturian2, Linlin Zhao3

  • 1Department of Cell, Molecular, and Developmental Biology, University of California, Riverside, CA 92521, USA.

International Journal of Molecular Sciences
|July 12, 2025
PubMed
Summary

This study developed two new models for mitochondrial DNA damage in trabecular meshwork cells, crucial for understanding open-angle glaucoma. These models revealed that mitochondrial damage is linked to increased expression of glaucoma-associated genes.

Keywords:
glaucomamitochondriatrabecular meshwork

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Area of Science:

  • Cell Biology
  • Ophthalmology
  • Genetics

Background:

  • Mitochondrial DNA (mtDNA) damage is observed in trabecular meshwork (TM) cells of patients with open-angle glaucoma (OAG).
  • Existing in vitro models for OAG do not adequately incorporate mtDNA damage, limiting their translational relevance.

Purpose of the Study:

  • To validate two novel methods for inducing and assessing mtDNA damage in immortalized TM cells.
  • To investigate the association between experimentally induced mtDNA damage and the expression of OAG-associated genes in TM cells.

Main Methods:

  • Established two models of mtDNA depletion in TM-1 cells: ethidium bromide (EtBr) treatment and doxycycline (Dox)-inducible expression of a mutant Uracil DNA Glycosylase 1 (UNG1 Y147A).
  • Quantified mtDNA levels and mRNA expression of OAG-associated transcripts (CTGF, FN1, PAI1, SFRP1) using qPCR.
  • Assessed mitochondrial protein levels (ATP5F1A, COXII, COXIV) via western blot.

Main Results:

  • Both EtBr treatment and Dox induction significantly decreased mtDNA levels per cell.
  • Significant reductions in COXII and COXIV protein levels were observed in both models.
  • Both models led to the upregulation of CTGF, FN1, and PAI1 mRNA; EtBr also upregulated SFRP1.

Conclusions:

  • Successfully validated two distinct in vitro models for inducing mitochondrial depletion in immortalized TM cells.
  • Demonstrated that mitochondrial damage in TM cells is associated with the upregulation of key OAG-associated transcripts.
  • These findings support a critical link between mitochondrial dysfunction and the pathogenesis of glaucoma.