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N. Fatma, E. Kubo, Y. Akagi, N. Agarwal, W. B. Thoreson, C. B. Camras, D. P. Singh; Transduction and Protective Efficacy of Biologically Active TAT-HA-PRDX6: Prevention of Retinal Ganglion Cells From TNF- and Glutamate-Induced Cytotoxicity. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4368.
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© ARVO (1962-2015); The Authors (2016-present)
The death of retinal ganglion cells (RGCs) in glaucoma has been associated with higher levels of reactive oxygen species (ROS) induced by locally increased glutamate and TNF-α and reduced expression of antioxidants within the cellular microenvironment of RGCs during intraocular pressure (IOP). We hypothesized that supplying a natural antioxidant peroxiredoxin 6 (PRDX6), should promote RGC survival. Using rat RGC-5 as a model, we investigated TNF-α and glutamate-induced ROS- mediated RGC death, and assessed the ability of PRDX6 fused to TAT transduction domain in attenuating TNFα-or glutamate-induced RGC death.
A full length of PRDX6 cDNA was cloned into Nco1 and EcoR1 sites of pTAT-HA prokaryotic vector, and protein was purified. Immunohistochemical staining of rat retina was done using antibody specific to PRDX6. RGCs were maintained in complete DMEM, and were cultured in twenty four well plate and treated with variable concentrations of TNF-α (10-100ng/ml) or glutamate (2-10mM) for variable times to assess their cytotoxicity. In parallel experiment, cultured cells were cotreated with variable concentrations of TAT-HA-PRDX6 protein (1-5ug/ml) to evaluate its protective efficacy. Western analysis and real-time quantitative PCR were used to assess endogenous PRDXs levels, and to confirm internalization of TAT-HA-PRDX6 protein. Biochemical assays: MTS and Trypan blue assays to assess cell viability; TUNEL and DAPI staining to define cell death; H2DCFH-DA dye to monitor ROS levels; and HIV-1LTR-CAT constructs to monitor NF-kB activation, were used to define TNFα- or glutamate-induced changes in RGCs.
PRDX6 was strongly expressed in RGCs, and levels of PRDX6 mRNA and protein expression were higher in RGCs compared to other members of the PRDX family. The TAT-HA-PRDX6 protein was transduced efficiently into RGCs, and was biologically active. RGCs exposed to glutamate and TNF-α showed elevated levels of ROS and reduced expression of PRDX6; these cells underwent apoptosis. Addition of PRDX6 protected RGCs from glutamate- and TNF-α-induced cytotoxicity, and inhibited NF-kB activity.
A supply of PRDX6 to RGCs protects the RGCs by blocking glutamate- and TNFα-induced ROS-mediated deleterious effects. Studies may provide foundation for rational use of antioxidant based therapeutics for treating/preventing RGCs death.
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