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A. J. Ondricek, N. Agarwal; Signaling Mechanisms of Mitochondria Associated Rat Retinal Ganglion Cell Death in Optic Neuropathies. Invest. Ophthalmol. Vis. Sci. 2007;48(13):242.
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© ARVO (1962-2015); The Authors (2016-present)
Optic neuropathies are a group of ocular diseases that progress as a gradual neurodegeneration culminating in vision loss due to the eventual death of retinal ganglion cells (RGCs.) There is evidence implicating oxidative stress in many neurodegenerative diseases. We hypothesize that mitochondrial distress results in retinal ganglion cell death in optic neuropathies and that Iodoacetic acid (IAA) inhibits ATP generation resulting in mitochondrial dysfunction and generation of ROS.
Transformed rat retinal ganglion cells (RGC-5) were treated with varying concentrations of IAA from 2 to 10 µM for a period of 24 hours. Antioxidants N-Acetyl Cysteine (NAC) (2mM) and Thiourea (10mM) were administered along with IAA. Cell viability was assessed by the neutral red dye uptake assay and/or by Calcein AM assay. Morphological changes were assessed by microscopy. Changes in Mitochondrial Membrane Potential (MMP) were determined by JC-1 fluorescent dye with an argon laser on a Zeiss LSM confocal microscope. Erk phosphorylation was determined by western blot analysis using Phospho-p44 and p42 antibody. Involvement of reactive oxygen species (ROS) was determined by H2DCF (dichlorofluorescein)-DA assay, and caspase-3 by fluorescent assay.
There was a dose dependent loss of cell viability by IAA treatment (2-10 µM) of RGC-5 cells. At a 6µM concentration in solution, IAA reduced cell viability by 50%. The antioxidants N-Acetyl Cysteine (2mM) and Thiourea (10mM) rescued RGC-5 cells from IAA induced cell death. IAA induces caspase activation in a dose dependent manner. IAA does not induce the activation of caspases in the presence of exogenous antioxidants NAC and Thiourea. IAA reduced the mitochondrial membrane potential in RGC-5 cells, which was rescued by NAC. NAC induces the phosphorylation of ERK 1 and 2.
IAA is cytotoxic to retinal ganglion cells. IAA does not induce death in retinal ganglion cells in the presence of exogenous antioxidants, N-acetyl cysteine and Thiourea. IAA induced RGC cell death involves oxidative stress that can be reversed by antioxidants. IAA creates a loss of MMP while NAC maintains the MMP. IAA does not result in phosphorylation of ERK1 and 2. NAC induces the phosphorylation of ERK1 and 2 in a time dependent manner while thiourea does not. NAC upregulates the expression of Bcl-2 in RGC-5 cells. Taken together, these results suggest IAA induced mitochondrial cytotoxicity involves Erk pathway.
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