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D. Vollrath, R. Zhang; Anti-Oxidant Protects Cells from Mutant Myocilin-Induced Apoptosis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4074.
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© ARVO (1962-2015); The Authors (2016-present)
To define the cellular response to expression of mutant myocilin and mitigate the resulting deleterious effects.
We created stable Chinese hamster ovary cell lines that express wild-type or mutant (P370L) myocilins under control of a tetracycline inducible promoter. Following induction of myocilin expression, we assessed the kinetics of activation of various indicators of cell stress including the unfolded protein response (UPR), reactive oxygen species, and apoptotic markers.
Within 48 hrs of induction, mutant myocilin forms intracellular aggregates that co-localize with endoplasmic reticulum proteins, as has been shown for other cell types. Expression of mutant myocilin induces 1) XBP1 mRNA splicing, which peaks by ~24 hours, indicating rapid activation of the IRE1 arm of the UPR, 2) reactive oxygen species accumulation by 24 hrs, as assessed by dichloro-dihydrofluorosceine diacetate staining, 3) phosphorylation of eIF2 (p-eIF2), demonstrating activation of the PERK arm of the UPR, 4) BiP, a UPR target gene, by more than 10-fold with a peak of mRNA at ~36 hrs, 5) CHOP, a target of the ATF6 and PERK arms of the UPR, with biphasic kinetics (peaks at 12 hrs and 60 hrs), 6) activation of JNK, initiator caspases 9 and 12, and executioner caspases 3 and 7 by 48 hrs. These effects were greatly diminished or absent in cells expressing wild-type myocilin and vector only controls done in parallel. Pre-treatment of cells with N-acetyl-cysteine (NAC), a precursor of glutathione, resulted in substantial reduction in mutant myocilin-induced reactive oxygen species. Interestingly, NAC treatment also blunts the mutant myocilin-induced UPR, as evidenced by diminished induction of BiP and p-eIF2. Importantly, NAC protects cells expressing mutant myocilin from apoptosis.
Expression of mutant myocilin results in generation of reactive oxygen species, robust activation of multiple arms of the UPR, and, ultimately, apoptosis. Augmentation of cellular anti-oxidant defenses causes a diminished UPR and mitigates myocilin-induced cell death. These results suggest NAC as a potential therapy for myocilin-associated glaucoma.
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