Pro370Leu mutant myocilin impairs mitochondrial functions in human trabecular meshwork cells

摘要

Purpose: Oxidative stress is a riskfactor for the onset and progression of primary open-angle glaucoma(POAG), but the exact molecular basis remains unknown. Here, weinvestigated the mechanisms for Pro370Leu mutant myocilin to inducemitochondrial dysfunction and subsequent reactive oxygen species (ROS)generation in trabecular meshwork (TM) cells obtained from POAGindividuals. Methods: Primary non-diseased human TMcultures were transfected with pIRES-EGFP (Mock), pIRES-wild-type (WT),or pIRES-Pro370Leu mutant myocilin. Transfection efficiency andmyocilin subcellular localization were determined by polymerase chainreaction (PCR), western blot analysis, and confocal microscopy. ROSlevels as well as free Ca2+ concentrations in cytoplasm ([Ca2+]c)and mitochondria ([Ca2+]m) were examined by2’7’-dichlorofluorescein diacetate (H2-DCF-DA), Fluo-3acetoxymethyl ester (Fluo-3/AM), and Rhod-2 acetoxymethyl ester(rhod-2/AM), respectively, using flow cytometry. Mitochondrialfunctions were revealed by changes in mitochondrial membrane potential(ΔΨm) and ATP production, which were found by fluorescent probe5,5′,6,6’-tetrachloro-1,1’3,3′-tetraethylbenzimid azolocarbocyanineiodide (JC-1) and a luciferin/luciferase-based ATP assay, respectively.Results: Both WT and Pro370Leu mutantmyocilin are localized in the mitochondria of TM cells as indicatedusing confocal microscopy and western blot analysis. Overexpression ofWT myocilin decreases ΔΨm, which is further reduced by Pro370Leu mutantmyocilin. TM cells that overexpressed Pro370Leu mutant myocilin havegreater cell death, higher endogenous ROS, [Ca2+]c,and [Ca2+]m levels, and lower ATP production, andyet, these effects are not seen in the overexpression of WT myocilin. Conclusions: Our findings suggested thatPro370Leu mutant myocilin causes mitochondrial defects, which may leadto TM cell dysfunction and even cell death. Therefore, preventivemeasures targeting mitochondrial protection may delay the onset ofglaucoma in individuals carrying the Pro370Leu myocilin mutation.