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

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.