May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Elevated Hydrostatic Pressure Triggers Release of OPA1 and Cytochrome c and Induces Apoptotic Cell Death in Differentiated RGC-5 Cells
Author Affiliations & Notes
  • W.-K. Ju
    Ophthalmology, Hamilton Glaucoma Center, La Jolla, California
  • K.-Y. Kim
    National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California
  • M. Angert
    Ophthalmology, Hamilton Glaucoma Center, La Jolla, California
  • A. Patel
    Ophthalmology, Hamilton Glaucoma Center, La Jolla, California
  • J. G. Crowston
    Ophthalmology, University of Melbourne, La Jolla, California
  • J. D. Lindsey
    Ophthalmology, Hamilton Glaucoma Center, La Jolla, California
  • M. H. Ellisman
    National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California
  • G. A. Perkins
    National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California
  • R. N. Weinreb
    Ophthalmology, Hamilton Glaucoma Center, La Jolla, California
  • Footnotes
    Commercial Relationships W. Ju, None; K. Kim, None; M. Angert, None; A. Patel, None; J.G. Crowston, None; J.D. Lindsey, None; M.H. Ellisman, None; G.A. Perkins, None; R.N. Weinreb, None.
  • Footnotes
    Support Vision for Glaucoma (La Jolla); NIH/NEI Grant EY014661 (JDL); NCRR P41 RR004050 (MHE)
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 5551. doi:
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      W.-K. Ju, K.-Y. Kim, M. Angert, A. Patel, J. G. Crowston, J. D. Lindsey, M. H. Ellisman, G. A. Perkins, R. N. Weinreb; Elevated Hydrostatic Pressure Triggers Release of OPA1 and Cytochrome c and Induces Apoptotic Cell Death in Differentiated RGC-5 Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5551.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: Optic Atrophy Type 1 (OPA1) is a dynamin-related GTPase of the inner mitochondrial membrane that is mutated in dominant optic atrophy. The presence of the mutation causes a progressive degeneration of retinal ganglion cells (RGCs) and vision loss. The purpose of this study is to determine whether elevated hydrostatic pressure alters mitochondrial structure, triggers release of OPA1 or cytochrome c from mitochondria, alters OPA1 gene expression, or can directly induce apoptotic cell death in cultured retinal ganglion cells.

Methods:: RGC-5 cells differentiated with succinyl concanavalin A (50 µg/ml) were exposed to 30 mmHg for 3 days in a pressurized incubator. As a control, parallel differentiated RGC-5 cell cultures were incubated simultaneously in a conventional incubator. Live RGC-5 cells were then labeled with MitoTracker Red and mitochondrial morphology was assessed by fluorescence microscopy and electron microscopy. OPA1 mRNA was measured by real time PCR. OPA1 protein, cytochrome c release and caspase-3 activation were examined by immunocytochemistry and Western blot. Apoptotic cell death was measured by the TUNEL method.

Results:: Mitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small organelles, was triggered at 3 days following elevated hydrostatic pressure. Electron microscopy showed that elevated hydrostatic pressure induced abnormal cristae depletion at 3 days. In addition, the mean length of mitochondrial cross section was decreased from 845.0 ± 41.0 nm to 571.3 ± 22.7 nm in pressure-treated cells. Pressure treatment also induced release of OPA1 and cytochrome c to the cytoplasm. Elevated hydrostatic pressure for 3 days significantly decreased OPA1 gene expression in differentiated RGC-5 cells, but total OPA1 protein expression was not changed. Elevated hydrostatic pressure also activated caspase-3 and induced subsequent apoptotic cell death.

Conclusions:: Elevated hydrostatic pressure triggered mitochondrial fission, release of OPA1 and cytochrome c into the cytoplasm, abnormal cristae depletion and subsequent apoptotic cell death in differentiated RGC-5 cells. These results suggest sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria.

Keywords: mitochondria • ganglion cells • intraocular pressure 
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