title: Pro370Leu mutant myocilin impairs mitochondrial functions in human trabecular meshwork cells.
abstract: OBJECTIVE
  Oxidative stress is a risk factor for the onset and progression of primary open-angle glaucoma (POAG), but the exact molecular basis remains unknown. Here, we investigated the mechanisms for Pro370Leu mutant myocilin to induce mitochondrial dysfunction and subsequent reactive oxygen species (ROS) generation in trabecular meshwork (TM) cells obtained from POAG individuals.
  
  
  METHODS
  Primary non-diseased human TM cultures were transfected with pIRES-EGFP (Mock), pIRES-wild-type (WT), or pIRES-Pro370Leu mutant myocilin. Transfection efficiency and myocilin subcellular localization were determined by polymerase chain reaction (PCR), western blot analysis, and confocal microscopy. ROS levels as well as free Ca(2+) concentrations in cytoplasm ([Ca(2+)](c)) and mitochondria ([Ca(2+)]m) were examined by 2'7'-dichlorofluorescein diacetate (H(2)-DCF-DA), Fluo-3 acetoxymethyl ester (Fluo-3/AM), and Rhod-2 acetoxymethyl ester (rhod-2/AM), respectively, using flow cytometry. Mitochondrial functions were revealed by changes in mitochondrial membrane potential (DeltaPsim) and ATP production, which were found by fluorescent probe 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimid azolocarbocyanine iodide (JC-1) and a luciferin/luciferase-based ATP assay, respectively.
  
  
  RESULTS
  Both WT and Pro370Leu mutant myocilin are localized in the mitochondria of TM cells as indicated using confocal microscopy and western blot analysis. Overexpression of WT myocilin decreases DeltaPsim, which is further reduced by Pro370Leu mutant myocilin. TM cells that overexpressed Pro370Leu mutant myocilin have greater cell death, higher endogenous ROS, [Ca(2+)](c), and [Ca(2+)](m) levels, and lower ATP production, and yet, these effects are not seen in the overexpression of WT myocilin.
  
  
  CONCLUSIONS
  Our findings suggested that Pro370Leu mutant myocilin causes mitochondrial defects, which may lead to TM cell dysfunction and even cell death. Therefore, preventive measures targeting mitochondrial protection may delay the onset of glaucoma in individuals carrying the Pro370Leu myocilin mutation.
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: He
authors.forename: Kar Wah
authors.initials: KW
authors.lastname: Leung
authors.forename: Ye-Hong
authors.initials: YH
authors.lastname: Zhuo
authors.forename: Jian
authors.initials: J
authors.lastname: Ge
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D016923:Cell Death; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005902:Glaucoma, Open-Angle / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D009154:Mutation; D017382:Reactive Oxygen Species / Q000378:metabolism; D014129:Trabecular Meshwork / Q000378:metabolism*; D000099179:Myocilin
chemical_list: D000255:Adenosine Triphosphate; D002118:Calcium; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D017382:Reactive Oxygen Species; D000099179:Myocilin