File Download

There are no files associated with this item.

  Links for fulltext
     (May Require Subscription)
Supplementary

Article: Pro370Leu mutant myocilin impairs mitochondrial functions in human trabecular meshwork cells

TitlePro370Leu mutant myocilin impairs mitochondrial functions in human trabecular meshwork cells
Authors
Issue Date2009
PublisherMolecular Vision. The Journal's web site is located at http://www.molvis.org/molvis/
Citation
Molecular Vision, 2009, v. 15, p. 815-825 How to Cite?
AbstractPurpose: 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 Ca2+ concentrations in cytoplasm ([Ca2+]c) and mitochondria ([Ca2+]m) were examined by 2′7′-dichlorofluorescein diacetate (H2-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 (ΔΨm) 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 ΔΨm, which is further reduced by Pro370Leu mutant myocilin. TM cells that overexpressed Pro370Leu mutant myocilin have greater cell death, higher endogenous ROS, [Ca2+]c, and [Ca2+]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. © 2009 Molecular Vision.
Persistent Identifierhttp://hdl.handle.net/10722/169855
ISSN
2015 Impact Factor: 2.11
2015 SCImago Journal Rankings: 1.037
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorHe, Yen_HK
dc.contributor.authorLeung, KWen_HK
dc.contributor.authorZhuo, YHen_HK
dc.contributor.authorGe, Jen_HK
dc.date.accessioned2012-10-25T04:57:06Z-
dc.date.available2012-10-25T04:57:06Z-
dc.date.issued2009en_HK
dc.identifier.citationMolecular Vision, 2009, v. 15, p. 815-825en_HK
dc.identifier.issn1090-0535en_HK
dc.identifier.urihttp://hdl.handle.net/10722/169855-
dc.description.abstractPurpose: 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 Ca2+ concentrations in cytoplasm ([Ca2+]c) and mitochondria ([Ca2+]m) were examined by 2′7′-dichlorofluorescein diacetate (H2-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 (ΔΨm) 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 ΔΨm, which is further reduced by Pro370Leu mutant myocilin. TM cells that overexpressed Pro370Leu mutant myocilin have greater cell death, higher endogenous ROS, [Ca2+]c, and [Ca2+]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. © 2009 Molecular Vision.en_HK
dc.languageengen_US
dc.publisherMolecular Vision. The Journal's web site is located at http://www.molvis.org/molvis/en_HK
dc.relation.ispartofMolecular Visionen_HK
dc.subject.meshAdenosine Triphosphate - Metabolismen_US
dc.subject.meshCalcium - Metabolismen_US
dc.subject.meshCell Deathen_US
dc.subject.meshCells, Cultureden_US
dc.subject.meshCloning, Molecularen_US
dc.subject.meshCytoskeletal Proteins - Genetics - Metabolismen_US
dc.subject.meshEye Proteins - Genetics - Metabolismen_US
dc.subject.meshGlaucoma, Open-Angle - Geneticsen_US
dc.subject.meshGlycoproteins - Genetics - Metabolismen_US
dc.subject.meshHumansen_US
dc.subject.meshMembrane Potential, Mitochondrialen_US
dc.subject.meshMicroscopy, Confocalen_US
dc.subject.meshMitochondria - Metabolismen_US
dc.subject.meshMutationen_US
dc.subject.meshReactive Oxygen Species - Metabolismen_US
dc.subject.meshTrabecular Meshwork - Metabolismen_US
dc.titlePro370Leu mutant myocilin impairs mitochondrial functions in human trabecular meshwork cellsen_HK
dc.typeArticleen_HK
dc.identifier.emailLeung, KW: kwleung1@hku.hken_HK
dc.identifier.authorityLeung, KW=rp01674en_HK
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.pmid19390644-
dc.identifier.scopuseid_2-s2.0-65349166689en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-65349166689&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume15en_HK
dc.identifier.spage815en_HK
dc.identifier.epage825en_HK
dc.identifier.isiWOS:000266410800001-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridHe, Y=7404942872en_HK
dc.identifier.scopusauthoridLeung, KW=13106059300en_HK
dc.identifier.scopusauthoridZhuo, YH=7005221373en_HK
dc.identifier.scopusauthoridGe, J=25421653600en_HK

Export via OAI-PMH Interface in XML Formats


OR


Export to Other Non-XML Formats