Glaucoma Neurodegenerative and Inflammatory Pathway Components Linked to TNF-␣/TNFR1 Signaling in the Glaucomatous Human Retina Xiangjun Yang,1 Cheng Luo,1 Jian Cai,2 David W. Powell,3,4 Dahai Yu,5 Markus H. Kuehn,5 and Gu¨lgu¨n Tezel1,6 PURPOSE. This study aimed to determine retinal proteomic al- work motivating further research. (Invest Ophthalmol Vis Sci. terations in human glaucoma, with particular focus on links to 2011;52:8442–8454) DOI:10.1167/iovs.11-8152 TNF-␣/TNFR1 signaling. METHODS. Human retinal protein samples were obtained from he prevailing view is that glaucoma pathogenesis is multi- 20 donors with (n ϭ 10) or without (n ϭ 10) glaucoma. Tfactorial, with a complex interplay of elevated intraocular Alterations in protein expression were individually analyzed by pressure-induced events and genetic/epigenetic/aging-related quantitative LC-MS/MS. Quantitative Western blot analysis with susceptibility factors contributing to neurodegeneration. Glial cleavage or phosphorylation site-specific antibodies was used activation response and secondary inflammatory/autoimmune for data validation, and cellular localization of selected proteins processes are also regarded as continuous components of glau- was determined by immunohistochemical analysis of the retina comatous neurodegeneration. It is widely accepted that in an additional group of glaucomatous human donor eyes (n ϭ chronic activation of glial cells and accompanying increases in 38) and nonglaucomatous controls (n ϭ 30). the production of proinflammatory cytokines, primarily includ- ing TNF-␣, are hallmarks of inflammation/parainflammation in RESULTS. Upregulated retinal proteins in human glaucoma in- glaucomatous tissue, although a cause-effect relationship re- cluded a number of downstream adaptor/interacting proteins mains to be validated.1,2 TNF-␣, with beneficial and neurotoxic and protein kinases involved in TNF-␣/TNFR1 signaling. Bioin- effects in the central nervous system (CNS) along with key formatic analysis of the high-throughput data established ex- physiological functions in the maintenance of immune homeo- tended networks of diverse functional interactions with death- stasis, has been implicated in the pathogenesis of a wide promoting and survival-promoting pathways and mediation of spectrum of human neurodegenerative diseases. It is also in- immune response. Upregulated pathways included death re- creasingly evident that TNF-␣ through the binding of TNFR1, a ceptor-mediated caspase cascade, mitochondrial dysfunction, death receptor, exhibits important links to glial activation re- endoplasmic reticulum stress, calpains leading to apoptotic ␬ sponse, mediation of retinal ganglion cell (RGC) death, and cell death, NF- B and JAK/STAT pathways, and inflammasome- inflammatory processes during the neurodegenerative injury in assembly mediating inflammation. Interestingly, retinal expres- glaucoma.3 sion pattern of a regulator molecule, TNFAIP3, exhibited prom- Despite growing evidence that supports important roles of inent variability between individual samples, and methylation TNF-␣ in glaucomatous neurodegeneration, opposing conse- of cytosine nucleotides in the TNFAIP3 promoter was found to quences of TNF-␣ signaling make it difficult to exploit for be correlated with this variability among glaucomatous donors. neuroprotective strategies. Respecting the diverse bioactivi- CONCLUSIONS. Findings of this study reveal a number of proteins ties of this multifunctional cytokine, molecular dissection of upregulated in the glaucomatous human retina that exhibit specific signaling components can provide the possibility to many links to TNF-␣/TNFR1 signaling. By highlighting various specifically inhibit RGC death or modulate immune response signaling molecules and regulators involved in cell death and without compromising survival-promoting signals. To better immune response pathways and by correlating proteomic find- understand molecular components of the neurodegenera- ings with epigenetic alterations, these findings provide a frame- tive signaling in human glaucoma, this study analyzed retinal protein samples obtained from donor eyes with or without glaucoma. Findings of this comparative analysis supported a prominent upregulation of TNF-␣/TNFR1 signaling in the From the Departments of 1Ophthalmology and Visual Sciences, 2 3 4 glaucomatous human retina. By highlighting various signaling Pharmacology and Toxicology, Medicine, Biochemistry & Molecular molecules and regulators involved in cell death and immune Biology, and 6Anatomical Sciences & Neurobiology, University of Lou- isville School of Medicine, Louisville, Kentucky; and 5Department of response pathways in human glaucoma, these findings provide Ophthalmology & Visual Sciences, University of Iowa, Iowa City, Iowa. framework information and motivate further research. Supported in part by National Eye Institute Grants R01 EY013813, R01 EY017131 (GT), and EY019485 (MHK), The Robert W. Rounsavall, Jr. Family Foundation, Inc. (GT), and by an unrestricted grant from MATERIALS AND METHODS Research to Prevent Blindness Inc. (Department of Ophthalmology and Visual Sciences). Donor Eyes Submitted for publication July 1, 2011; revised August 24 and Retinal protein samples obtained from 10 human donor eyes with September 7, 2011; accepted September 8, 2011. glaucoma (age, 84.7 Ϯ 8) and 10 eyes without glaucoma (age, 83.7 Ϯ Disclosure: X. Yang, None; C. Luo, None; J. Cai, None; D.W. Powell, None; D. Yu, None; M.H. Kuehn, None; G. Tezel, None 7) were individually analyzed by capillary liquid chromatography cou- Corresponding author: Gu¨lgu¨n Tezel, University of Louisville pled with linear ion trap mass spectrometry (LC-MS/MS). As previously School of Medicine, Kentucky Lions Eye Center, 301 E. Muhammad Ali described,4,5 retinal tissue punches were collected within Ͻ6 hours Boulevard, Louisville, KY 40202; [email protected]. after death, and glaucomatous eyes were well documented. Investigative Ophthalmology & Visual Science, October 2011, Vol. 52, No. 11 8442 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. Downloaded from iovs.arvojournals.org on 09/24/2021 IOVS, October 2011, Vol. 52, No. 11 TNF-␣/TNFR1 Signaling in the Glaucomatous Human Retina 8443 In addition, cellular localization of selected proteins was deter- tems). Canonical pathway analysis identified the pathways from the IPA mined by immunohistochemical analysis of retinal tissue sections ob- library that were most significant to the dataset by the right-tailed Fisher’s tained from an additional group of glaucomatous and nonglaucoma- exact test. tous human donor eyes. This group included 38 donor eyes with a diagnosis of glaucoma (age, 76.8 Ϯ 11) and 30 eyes without glaucoma Western Blot Analysis (age, 71.0 Ϯ 15), all fixed within 12 hours after death. Detailed information on donor demographics and clinical data has been previ- Immunoblotting used primary antibodies to cleaved caspase 1 (1: ously published.6 All the human donor eyes were handled according to 1000; Millipore, Billerica, MA), caspases 3 and 8 (1:1000; Cell the tenets of the Declaration of Helsinki. Signaling, Danvers, MA), caspases 9 and 12 (1:500; Abcam, Cam- bridge, MA), nuclear factor kappa B (NF-␬B) subunits, p50 [phos- Proteomic Analysis pho-Ser932] (1:500; GenWay, San Diego, CA) and p65 [phospho- Ser276] (1:1000; Cell Signaling), signal transducers and activators of Protein samples prepared with a lysis buffer containing 50 mM Hepes- transcription (STAT)1 [phospho-Tyr701], STAT2 [phospho-Tyr690], KOH pH 8.0, 100 mM KCl, 2 mM EDTA, 0.10% NP-40, 2 mM dithio- STAT3 [phospho-Ser727], STAT4 [phospho-Tyr693], STAT5 [phos- threitol, 10% glycerol, and protease and phosphatase inhibitors were pho-Tyr694], STAT6 [phospho-Tyr641], (1:1000; Cell Signaling), and analyzed by label-free quantitative LC-MS/MS, as previously described.7 TNF-␣–induced protein 3 (TNFAIP3; 1:1000; Epitomics, Burlingame, Briefly, trypsin-digested samples were loaded onto an analytical 2D CA). In addition, a ␤-actin antibody (Sigma-Aldrich, St. Louis, MO) was capillary chromatography column packed with strong cation exchange used to reprobe immunoblots for loading and transfer control. The (SCX) and C reversed-phase (RP) resin (Phenomenex, Torrance, CA). 18 secondary antibody incubation used a specific IgG conjugated with This biphasic column was attached to an analytical RP chromatography horseradish peroxidase (1:2000; Sigma-Aldrich). The primary antibody column with an integrated, laser-pulled emitter tip. Peptides were was omitted to provide negative control. After normalization to ␤-actin, eluted from SCX with seven-step gradients of 5%, 10%, 15%, 30%, 50%, the average band intensity value obtained from nonglaucomatous sam- 70%, and 100% of 500 mM ammonium acetate and eluted into a linear ples was used to calculate the fold change in protein expression in ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) glaucomatous samples. according to a linear HPLC gradient (20-minute 0% B, 80-minute 40% B, and 90-minute 60% B at a flow rate of 200 nL/min with mobile phase-A 5% acetonitrile/0.1% formic acid and mobile phase-B 80% acetonitrile/ Morphologic Analysis 0.1% formic acid). Protein identification from MS/MS spectra was Double immunofluorescence labeling used the same primary antibod- performed with proteomics analysis software (Sequest Sorcerer; ies described for Western blot analysis (1:100). In addition, antibodies Sage-N Research, San Jose, CA), which was set up to search a FASTA