Kennedy Shane P, Tsaturian Emily, Zhao Linlin, Morgan Joshua T
Department of Cell, Molecular, and Developmental Biology, University of California, Riverside, CA 92521, USA.
Department of Bioengineering, University of California, Riverside, CA 92521, USA.
Int J Mol Sci. 2025 Jun 28;26(13):6255. doi: 10.3390/ijms26136255.
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.
小梁网（TM）细胞中的线粒体DNA（mtDNA）损伤发生在开角型青光眼（OAG）中。然而，目前用于模拟TM细胞中OAG样变化的体外模型并未明确纳入mtDNA损伤。这项工作验证了永生化TM细胞中两种mtDNA损伤方法，并评估了与OAG相关的表达变化。通过溴化乙锭（EtBr）处理和强力霉素（Dox）诱导TM-1细胞中的尿嘧啶DNA糖基化酶1（UNG1）突变体（Y147A）版本（TM-1），使TM-1细胞中的mtDNA耗竭。通过蛋白质印迹法测量线粒体蛋白（ATP5F1A、COXII和COXIV）的水平。通过qPCR测量mtDNA水平以及与OAG相关转录本（、、和）的mRNA。在EtBr处理的TM-1细胞和诱导的TM-1细胞中，所有处理时间点每个细胞的mtDNA水平均有统计学意义的显著下降。ATP5F1A的蛋白水平没有显著变化；EtBr和Dox诱导后，COXII和COXIV均显示出显著下降。两种模型均导致、和上调；此外，EtBr处理而非Dox诱导导致上调。总之，在永生化TM细胞中证明了两种线粒体耗竭模型；损伤与OAG相关转录本的增加有关，支持线粒体损伤与青光眼表型之间的联系。
