The transcription factor c-jun is activated in retinal ganglion cells in experimental rat glaucoma * Hana Levkovitch-Verbin a, * , Harry A. Quigley b , Keith R.G. Martin b , Noga Harizman a , Danielle F. Valenta b , Mary Ellen Pease b , Shlomo Melamed a a Sam Rothberg Molecular Biology Lab, Goldschleger Eye Institute, Sheba Medical Center, Tel-Aviv University, Tel-Hashomer 52621, Israel b Glaucoma Research Laboratory, Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA Received 18 May 2004; accepted in revised form 29 November 2004 Available online 4 January 2005 Abstract This study investigates the role of the MAP kinase pathway including c-jun, ATF-2 and JNK in glaucomatous eyes of rats and in optic nerve transection. Glaucoma was induced in one eye of 51 adult Wistar rats by laser treatment to the trabecular meshwork. Eighteen further rats underwent unilateral optic nerve transection. We studied the transcription factor c-jun, its activated form, phospho-c-jun, the transcription factor p-ATF-2, and the enzyme JNK by immunohistochemistry. The activation of p-c-jun was also investigated using western blot analysis. Treated and control eyes were compared in a masked way at multiple time points after injury. We found a statistically significant increase in immunolabelling for c-jun and phospho-c-jun in retinal ganglion cells (RGCs) from 1 day to 4 weeks after intraocular pressure (IOP) elevation. At 1 and 2 days after the laser treatment, a mean of 2.9G3.3 RGCs mm K1 were positive for c-jun (nZ12, pZ0.005, t-test), increasing to a mean of 13.4G7.5 cells mm K1 at 1 week (nZ18, pZ0.00005), and decreasing to 2.3G2.0 cells mm K1 at 2 weeks (nZ5, pZ0.04) and 0.1G0.1 cells mm K1 at 2 months. Few of the 47 control eyes had any labelling for c- jun or phospho-c-jun, while between 80 and 100% of elevated IOP eyes showed positivity during the first 2 weeks of experimental glaucoma. After optic nerve transection, c-jun and phospho-c-jun were also significantly activated at 1, 2 and 9 days (p!0.03, t-test). Western blot analysis demonstrated significantly increased phospho-c-jun amounts in both transected and glaucomatous eyes compared to control fellow eyes 1 week following treatment. JNK was not significantly activated in glaucoma or optic nerve transection. P-ATF-2 was not significantly activated in glaucoma, but was significantly increased 2 days after optic nerve transection. We conclude that the process leading to RGC death in experimental glaucoma and after optic nerve transection involves the activation of c-jun at the RGC layer. C-jun is activated more gradually in glaucoma then after optic nerve transection. q 2004 Elsevier Ltd. All rights reserved. Keywords: glaucoma; optic nerve transection; retinal ganglion cells; c-jun; JNK 1. Introduction RGCs die by apoptosis in glaucomatous eyes of humans, monkeys and rats (Garcia-Valenzuela et al., 1995; Quigley et al., 1995; Kerrigan et al., 1997; Nickells, 1999). Neurotrophin deprivation may be an important factor in RGC death in glaucoma, as a result of obstructed retrograde axonal transport to RGCs from their target cells in the brain (Johnson et al., 2000; Pease et al., 2000; Quigley et al., 2000). Brain derived neurotrophic factor (BDNF) has a neuroprotective effect for RGCs in vitro and in several disease models, including glaucoma (Johnson et al., 1986; Di Polo et al., 1998; Klocker et al., 2000; Ko et al., 2000, 2001). Neurotrophin withdrawal may lead to apoptosis through gene activation by transcription factors, including members of the mitogen activated protein kinase (MAPK) pathway (Mesner et al., 1995; Seger and Krebs, 1995; Weng et al., 1999; Yuan and Yankner 2000; Chang and Karin, 0014-4835/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2004.11.016 Experimental Eye Research 80 (2005) 663–670 www.elsevier.com/locate/yexer * Supported in part by PHS Research Grants EY 02120 (Dr Quigley), EY 01765 (Core Facility Grant, Wilmer Institute), by the Glaucoma Research Foundation, San Francisco, CA, by the David Worthen Fellowship and by the Claire and Amedee Maratier Institute, Tel-Aviv University, Israel. * Corresponding author. Dr Hana Levkovitch-Verbin, Sam Rothberg Molecular Biology Lab, Goldschleger Eye Institute, Sheba Medical Center, Tel-Aviv University, Tel-Hashomer 52621, Israel. E-mail address: halevko@hotmail.com (H. Levkovitch-Verbin).