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Galina Dvoriantchikova, Jeff Grant, Andrea Rachelle C. Santos, Eleut P. Hernandez, Valery I. Shestopalov, Dmitry V. Ivanov; Expression of Nox Family Proteins In Normal and Ischemic RGCs. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5328.
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© ARVO (1962-2015); The Authors (2016-present)
Increasing evidence suggests that NADPH oxidase complexes are an important source of cellular ROS and oxidative stress-mediated damage after ischemia/reperfusion (IR) injury in neurons. This study was designed to investigate superoxide production and the expression of the Nox family of NADPH oxidase proteins in normal and ischemic RGCs.
We evaluated the in vivo and in vitro expression levels of members of the Nox family and related subunits in normal RGCs and after ischemia by quantitative RT-PCR. IR injury was induced by unilateral elevation of intraocular pressure via direct corneal canulation. RGCs isolated by immunopanning from retinas were exposed to oxygen and glucose deprivation (OGD). Corresponding changes in protein abundances in RGCs were analyzed by immunohistochemistry and immunocytochemistry. The level of ROS generated was assayed by dihydroethidium (DHE). The Nox inhibitors VAS2870, AEBSF and ML090 were then tested to determine if it altered the production of ROS within the RGCs.
We report that RGCs produced ROS after OGD. The Nox inhibitors VAS2870, AEBSF and ML090 decreased the burst stimulated by the OGD. We found that RGCs express catalytic Nox1, Nox2, Nox4, Duox1, as well as regulatory Ncf1/p47phox, Ncf2/p67phox, Cyba/p22phox, Noxo1, Noxa1 subunits and Rac1, Rac2 and Rac3 under normal conditions and after ischemia. However, RGCs express only low levels of catalytic Nox2, Nox4 and Duox1, and regulatory Ncf1/p47, Ncf2/p67 subunits, but exhibit significantly higher level of catalytic subunit Nox1 and subunits required for optimal activity of Nox1, including Cyba/p22phox, Noxo1, and Rac1. The level of catalytic Nox3 and Duox2, and regulatory Ncf4/p40phox subunits were statistically insignificant in RGCs. Importantly, OGD treatment increased the expression level of Nox1, while the levels of Nox2 and Nox4 were reduced after OGD
Our study showed that catalytic subunits Nox1, Nox 2, Nox4, Duox1 and related regulatory subunits are present in normal and ischemic RGCs. The expression of proteins of the Nox family in RGCs suggests that NADPH-oxidase related production of ROS can play a role in normal RGC function, but excessive Nox-derived ROS production after IR can mediate RGC death. Importantly, high levels of catalytic subunit Nox1 and subunits required for optimal activity of Nox1 in RGCs suggest that this enzyme could be a major source of ROS in ischemic RGCs produced by NADPH oxidases.
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