September 2015
Volume 56, Issue 10
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Glaucoma  |   September 2015
Proteomics Analysis of Molecular Risk Factors in the Ocular Hypertensive Human Retina
Author Affiliations & Notes
  • Xiangjun Yang
    Department of Ophthalmology Columbia University College of Physicians and Surgeons, New York, New York, United States
  • Gözde Hondur
    Department of Ophthalmology Columbia University College of Physicians and Surgeons, New York, New York, United States
  • Ming Li
    Department of Medicine, University of Louisville School of Medicine, Louisville, Kentucky, United States
  • Jian Cai
    Department of Medicine, University of Louisville School of Medicine, Louisville, Kentucky, United States
  • Jon B. Klein
    Department of Medicine, University of Louisville School of Medicine, Louisville, Kentucky, United States
    Robley Rex Veterans Administration Medical Center, Louisville, Kentucky, United States
  • Markus H. Kuehn
    Department of Ophthalmology & Visual Sciences, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States
  • Gülgün Tezel
    Department of Ophthalmology Columbia University College of Physicians and Surgeons, New York, New York, United States
  • Correspondence: Gülgün Tezel, Edward S. Harkness Eye Institute, Columbia University Medical Center, 635 West 165th Street, Box 102, New York, NY 10032, USA; gt2320@columbia.edu
Investigative Ophthalmology & Visual Science September 2015, Vol.56, 5816-5830. doi:10.1167/iovs.15-17294
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      Xiangjun Yang, Gözde Hondur, Ming Li, Jian Cai, Jon B. Klein, Markus H. Kuehn, Gülgün Tezel; Proteomics Analysis of Molecular Risk Factors in the Ocular Hypertensive Human Retina. Invest. Ophthalmol. Vis. Sci. 2015;56(10):5816-5830. doi: 10.1167/iovs.15-17294.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To better understand ocular hypertension–induced early molecular alterations that may determine the initiation of neurodegeneration in human glaucoma, this study analyzed retinal proteomic alterations in the ocular hypertensive human retina.

Methods: Retina samples were obtained from six human donors with ocular hypertension (without glaucomatous injury) and six age- and sex-matched normotensive controls. Retinal proteins were analyzed by two-dimensional LC-MS/MS (liquid chromatography and linear ion trap mass spectrometry) using oxygen isotope labeling for relative quantification of protein expression. Proteomics data were validated by Western blot and immunohistochemical analyses of selected proteins.

Results: Out of over 2000 retinal proteins quantified, hundreds exhibited over 2-fold increased or decreased expression in ocular hypertensive samples relative to normotensive controls. Bioinformatics linked the proteomics datasets to various pathways important for maintenance of cellular homeostasis in the ocular hypertensive retina. Upregulated proteins included various heat shock proteins, ubiquitin proteasome pathway components, antioxidants, and DNA repair enzymes, while many proteins involved in mitochondrial oxidative phosphorylation exhibited downregulation in the ocular hypertensive retina. Despite the altered protein expression reflecting intrinsic adaptive/protective responses against mitochondrial energy failure, oxidative stress, and unfolded proteins, no alterations suggestive of an ongoing cell death process or neuroinflammation were detectable.

Conclusions: This study provides information about ocular hypertension–related molecular risk factors for glaucoma development. Molecular alterations detected in the ocular hypertensive human retina as opposed to previously detected alterations in human donor retinas with clinically manifest glaucoma suggest that proteome alterations determine the individual threshold to tolerate the ocular hypertension–induced tissue stress or convert to glaucomatous neurodegeneration when intrinsic adaptive/protective responses are overwhelmed.

Glaucoma, a leading cause of blindness, is a multifactorial neurodegenerative disease damaging the optic nerve, including retinal ganglion cell (RGC) somas, axons, and synapses. The optic nerve degeneration in glaucoma has been linked to intraocular pressure–generated mechanical and/or vascular stress,1,2 aging,3 genetic predispositions,4 epigenetic risk factors,5 compartmentalized subcellular processes,6 and secondary neurodegenerative events due to oxidative stress,7,8 glial activation/dysfunction,810 and glia-mediated inflammation.1013 A major goal of glaucoma research has been uncovering the molecular pathways of neurodegeneration to thereby develop new and improved treatment strategies for neuroprotection/rescue, neuroregeneration, and immunomodulation in patients with glaucoma. Proteomics analysis techniques offer an important toolbox for accomplishing this research aim. Indeed, many previous studies analyzing the proteomic alterations in human glaucoma and animal models have provided important insights into molecular pathways of glaucomatous neurodegeneration.14,15 
While glaucoma refers to patients with clinical characteristics of optic nerve injury that can be assessed by structural and functional analysis techniques, in a group of patients, intraocular pressure is found elevated (>21 mm Hg) with no detectable damage to the optic nerve. These individuals who are at increased risk for developing glaucoma are referred to as ocular hypertensives. Based on the multicenter clinical trial by the Ocular Hypertension Treatment Study group, 9.5% of these patients with ocular hypertension convert to glaucoma over 5 years if their high intraocular pressures are untreated, while 4.4% among the patients treated to lower intraocular pressure develop glaucoma.16,17 Clinical risk factors for glaucoma development are well studied; however, molecular understanding of human glaucoma is limited. 
The complexity of glaucomatous neurodegeneration that involves multiple molecular pathways for primary injury increases even further with a range of secondary injury processes. Undoubtedly, early molecular alterations, as opposed to secondary molecular responses, are more critical to determine the molecular mechanisms underlying the initiation of glaucomatous neurodegeneration. To gain information about ocular hypertension–related early molecular alterations, this study analyzed retinal protein samples from ocular hypertensive human donors. Herein, we present our retinal proteomics data from six human donors with ocular hypertension in comparison to data from age- and sex-matched ocular normotensive controls (as well as by considering the previous retinal proteomics data from glaucomatous human donors1821). The presented data reflect ocular hypertension–related “molecular risk factors,” the accumulation of which has potential to distress the physiological equilibrium toward glaucoma development. 
Methods
Donor Eyes
Human retinal protein samples were obtained from six donors with ocular hypertension (four females aged 82, 85, 86, and 89 years; two males aged 83 and 87 years) and six control donors without ocular hypertension or glaucoma (four females aged 80, 83, 87, and 88 years; two males aged 84 and 85 years). Ocular hypertension was diagnosed based on elevated intraocular pressure readings (>21 mm Hg; range, 22–28 mm Hg) on multiple occasions through the clinical follow-up for up to over 10 years with no detectable optic disc or visual field abnormalities characteristic of glaucoma. Ocular hypertensive donors did not receive any intraocular pressure-lowering treatment. Both ocular hypertensive and control donors had systemic treatment for health conditions, including cardiovascular disease or cancer. Control donors had no history of eye disease except for one control donor who was matched with one of the ocular hypertensive donors with aging-related macular degeneration (none of the retina samples included the macular region). 
As previously described,18,22 6-mm trephine punches of the retina were taken from each quadrant of the retina in a standardized manner and immediately frozen in liquid nitrogen. In addition, central-to-peripheral wedges of the retina were fixed for 3 hours in freshly prepared 4% paraformaldehyde in phosphate-buffered saline. The fixed tissues were then infiltrated with increasing concentrations of sucrose solution and embedded in optimal cutting temperature medium (Sakura Finetek USA, Inc., Torrance, CA, USA) for cryostat sectioning. All of the samples were collected within less than 6 hours after death. 
We analyzed retinal protein extracts by two-dimensional capillary liquid chromatography and linear ion trap mass spectrometry (LC-MS/MS) using oxygen isotope labeling for relative quantification of protein expression. This quantitative proteomics analysis technique compares signal intensities in the mixture of experimental sample (here, 18O-labeled ocular hypertensive retina) with control sample (16O-labeled ocular normotensive control retina) by mass spectrometry. For this analysis, we carefully paired our samples (six ocular hypertensive samples and six normotensive controls) by matching for donor age and sex (as well as the history of systemic or eye disease and the postmortem period till sample collection). Although the oxygen isotope labeling–based quantitative technique is based on the comparison of 1 ocular hypertensive sample with 1 age- and sex-matched normotensive control in each of the 6 sample pairs, we also analyzed all of these 12 samples individually (6 ocular hypertensive and 6 normotensive) by Western blotting for validation of protein expression for selected proteins. In addition, protein localization was analyzed in retinal tissue sections obtained from the same donors by immunohistochemistry. Tissue collection and handling adhered to the tenets of the Declaration of Helsinki. 
Proteome Analysis for Relative Quantification of Protein Expression
Retinal proteins were extracted with a lysis buffer containing 50 mM HEPES-KOH pH 8.0, 100 mM KCl, 2 mM EDTA, 0.10% NP-40, 2 mM dithiothreitol, 10% glycerol, and protease and phosphatase inhibitors as previously described.21,23 Trypsin-digested protein samples were analyzed by LC-MS/MS after isotope labeling (16O/18O labeling2426) as we previously described.18 To allow relative quantification of protein expression, ocular hypertensive samples were labeled with 18O and control samples with normal 16O. Briefly, protein lysates were desalted and dried after digestion with 0.4 μg/μL trypsin (Promega, Madison, WI, USA). For trypsin-catalyzed 16O-to-18O exchange/labeling, samples were dissolved in a solution containing 50 μL 50 mM Tris buffer (pH 7.8), 5 mM CaCl2, and 1 μg trypsin prepared in either normal or 18O-labeled water (Sigma-Aldrich Corp., St. Louis, MO, USA). Following their incubation at 37°C for 24 hours, the samples were heated to 100°C for 10 minutes and acidified with 25 μL 5% formic acid (FA). Each pair of ocular hypertensive and age- and sex-matched control samples was then mixed, and desalted with C18 spin column (Nest Group, Southborough, MA, USA). After fractionation by strong cation exchange (SCX) as previously described,19,20 the samples dissolved in 25 μL loading buffer were loaded onto a SCX cartridge and stepwise eluted with 20 μL 15 SCX buffers (containing ammonium acetate/acetic acid) of different ionic strength and pH. Eluted fractions were concentrated to approximately 2 μL by speedvac, mixed with 10 μL 5% acetonitrile (ACN)/0.1% FA, and analyzed by a nanoAcquity (Waters, Milford, MA, USA)-LTQ Orbitrap XL (Thermo Scientific, San Jose, CA, USA) system in data-dependent scan mode. An in-house packed capillary column (0.1 × 130-mm column packed with 3.6 μm, 200 Å Aeris XB-C18) and a solvent gradient with 0.1% FA and ACN/0.1% FA were used for separation. 
Data files from each pair of samples were searched against the human database by Proteome Discoverer v1.4 (Thermo Scientific). Protein identification was based on at least three high-confidence peptide matches (false discovery rate < 1%). Relative abundances for differentially labeled peptides were calculated from peak areas of their monoisotopic peaks and reported as heavy-to-light (18O/16O) ratios. The heavy-to-light ratio values were converted to the presented fold change values, where the negative inverse (−1/x) was taken for values between 0 and 1, so that a −x value presented in tables represent x-fold downregulation in protein expression. 
Similarly to our previous studies,21,23 we used a pathway analysis software (Ingenuity Pathway Analysis; Ingenuity Systems, Mountain View, CA, USA) for bioinformatics analysis of the LC-MS/MS results. The proteomics datasets and the corresponding expression values were uploaded into the application to search functional patterns from the Ingenuity Pathway Analysis library. Canonical pathway analysis identified the pathways from the Ingenuity library that were most significantly associated with our datasets by the right-tailed Fisher's exact test. 
Western Blot Analysis
For proteomics data validation, retinal protein samples were also analyzed by quantitative Western blot analysis for selected molecules. Immunoblotting followed the previously described protocols in principal.19,20 Briefly, the proteins separated by SDS-PAGE using precast polyacrylamide gels (Bio-Rad, Hercules, CA, USA) were electrophoretically transferred to a nitrocellulose membrane (Bio-Rad). After blocking with the Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 hour, we incubated the membranes with monoclonal antibodies for 60 minutes at room temperature with gentle shaking. The primary antibodies included those to heat shock cognate protein 71 (HSPA8; 1:500; Abcam, Cambridge, MA, USA) and superoxide dismutase 1 (SOD1; 1:500; Abcam). In addition, we used a cocktail of five mouse monoclonal primary antibodies against components of mitochondrial oxidative phosphorylation (1:200; Abcam), including complex I, nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 beta subcomplex 8 (NDUFB8); complex II, succinate dehydrogenase complex subunit B (SDHB); complex III, ubiquinol-cytochrome c reductase core protein II (UQCRC2); complex IV, cytochrome c oxidase subunit II (COX2); and complex V, adenosine triphosphate (ATP) synthase subunit alpha 1 (ATP5A1). Another primary antibody that we also used to probe the membranes was a phosphorylation site-specific (T231) antibody to microtubule-associated protein tau (MAPT, 1:1000; Abcam). The primary antibodies were mixed with a beta-actin antibody (1:500; Sigma-Aldrich Corp.) for loading and transfer control. The antibody dilutions used were optimum as assessed preliminarily by signal intensity, background staining, and amount of nonspecific detection with varying antibody concentrations. To lower background, 0.1% Tween-20 was added to the diluted antibody before incubation. After washing in phosphate-buffered saline containing 0.1% Tween-20, membranes were incubated with infrared IRDye (700- or 800-nm channel dye)-labeled secondary antibodies (1:10,000; LI-COR Biosciences). We diluted the fluorescently labeled secondary antibodies in Odyssey blocking buffer, and similar to primary antibodies, added Tween-20 to the diluted antibody. After incubation with a mixture of secondary antibodies for 60 minutes at room temperature, membranes were washed in phosphate-buffered saline. The membranes were then scanned in the appropriate channels for two-color detection using the Odyssey Infrared Imaging (LI-COR Biosciences). The linear auto scan function was used to automatically optimize scan intensity to generate an image without signal saturation. After background subtraction and normalization to beta-actin, the signal intensity obtained from normotensive control samples was used to calculate the fold change in protein expression in ocular hypertensive samples. 
Immunohistochemical Analysis
To determine the cellular localization and extent of selected proteins, histologic sections of the human donor retinas were also analyzed after specific immunolabeling as previously described.19,20 Immunofluorescence labeling utilized the same antibodies to HSPA8, SOD1, and phosphorylated tau (p-tau) as described above for immunoblotting (1:200–1:500). In addition, a polyclonal antibody against a neuronal marker protein, neuron-specific nuclear protein (NeuN; 1:200; Abcam), was used to determine protein localization by double immunolabeling. A mixture of Alexa Fluor 488- or 568-conjugated IgGs (1:500, Life Technologies, Waltham, MA, USA) was used for the secondary antibody incubation. DAPI (4′,6-diamidino-2-phenylindole; Thermo Fisher Scientific, Waltham, MA, USA) was used for nuclear counterstaining. Slides were examined by fluorescence microscopy, and images were recorded by digital photomicrography (Carl Zeiss, Thornwood, NY, USA). Negative controls were performed by replacing the primary antibody with serum or using an inappropriate secondary antibody to determine species specificity. 
Results
Quantitative Proteomics Analysis Indicated a Prominent Stress Response to Ocular Hypertension in the Human Retina
Retinal proteome analysis by LC-MS/MS after oxygen isotope labeling detected over 2000 proteins identified based on three or more identical peptides (with up to 85% sequence coverage). Based on relative quantification of protein expression, hundreds of these proteins were up- or downregulated in ocular hypertensive samples relative to their age- and sex-matched ocular normotensive controls. 
To initially provide overall information, Figure 1 presents the selected 30 canonical pathways that were significantly associated with our datasets by bioinformatics analysis. Bioinformatics knowledge-based analysis of the quantitative data by the Ingenuity Pathway Analysis system indicated that many of the retinal proteins exhibiting over 2-fold altered expression in ocular hypertensive samples relative to controls represented a prominent adaptive/protective molecular activity. Major molecular alterations were linked to molecular transport (including clathrin-mediated and caveolar-mediated endocytosis) and protein synthesis, cellular assembly, and organization (including eukaryotic initiation factors, eIF2 and eIF4, and p70S6K, a serine/threonine kinase targeting S6 ribosomal protein, p70S6K, involved in the initiation and regulation of translation). Major molecular alterations were also linked to cellular/neuronal function and maintenance, including cell adhesion, cell–cell interaction, and axonal guidance. The cell signaling pathways associated with our datasets included mechanistic target of rapamycin (mTOR), Rho family guanosine triphosphatase (GTPase), ephrin receptor, integrin, 14-3-3, cAMP response element-binding protein (CREB), extracellular signal–regulated kinase (ERK/MAPK), P2Y purigenic receptor, phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), calcium, neurotrophin/tyrosine kinase receptor (TRK), janus kinase and signal transducer and activator of transcription (JAK/STAT), and endothelin-1 signaling. As seen in Figure 1, the top canonical pathways that were significantly associated with our datasets also pointed to a prominent stress response related to mitochondrial dysfunction and endoplasmic reticulum stress that were characterized by upregulated expression of various proteins linked to production of nitric oxide and reactive oxygen species, nuclear factor-erythroid 2-related factor-2 (NRF2)-mediated oxidative stress response, protein ubiquitination and unfolded protein response, and DNA repair. 
Figure 1
 
Bioinformatics analysis of proteomics datasets. Retinal protein samples from ocular hypertensive human retinas and normotensive controls were analyzed by two-dimensional LC-MS/MS using oxygen isotope labeling for relative quantification of protein expression. Quantitative proteomics datasets were then searched for functional patterns by the Ingenuity Pathway Analysis. This analysis linked the ocular hypertension–related retinal proteomic alterations to various canonical pathways from the Ingenuity knowledge library, including those shown in this graph. The bars display the significance of these associations (the −log of P value calculated by Fisher's exact test right-tailed), and the orange squares connected by a thin line represent the ratio of the number of proteins in our dataset of a given pathway to the total number of proteins in this canonical pathway.
Figure 1
 
Bioinformatics analysis of proteomics datasets. Retinal protein samples from ocular hypertensive human retinas and normotensive controls were analyzed by two-dimensional LC-MS/MS using oxygen isotope labeling for relative quantification of protein expression. Quantitative proteomics datasets were then searched for functional patterns by the Ingenuity Pathway Analysis. This analysis linked the ocular hypertension–related retinal proteomic alterations to various canonical pathways from the Ingenuity knowledge library, including those shown in this graph. The bars display the significance of these associations (the −log of P value calculated by Fisher's exact test right-tailed), and the orange squares connected by a thin line represent the ratio of the number of proteins in our dataset of a given pathway to the total number of proteins in this canonical pathway.
Major Molecular Alterations in the Ocular Hypertensive Human Retina Were Linked to Unfolded Protein Response, Mitochondrial Energy Failure, and Oxidative Stress
To next provide more focused information, we present our most prominent and consistent data in three tables. Table 1 lists the proteins, including various chaperones and stress-response proteins involved in protein ubiquitination and unfolded protein response, and Table 2 lists various proteins involved in cellular redox homeostasis and oxidative stress response, many of which were upregulated in ocular hypertensive samples. However, as presented in Table 3, many proteins involved in mitochondrial oxidative phosphorylation exhibited over 2-fold downregulation in ocular hypertensive samples. To be able to present the overall protein regulation trend within the presented pathways, these three tables list all relevant proteins (not only those exhibiting over 2-fold increased or decreased expression) in six ocular hypertensive samples relative to normotensive controls. Apparently, the identified proteins included those with predicted protein locations within different cellular compartments, including cytoplasm, nucleus, and plasma membrane. In addition to number of peptides and percentage of peptide coverage presented in these tables, Supplementary Material for each table includes peptide sequence coverage maps for selected proteins. 
Table 1
 
Proteins Linked to Protein Ubiquitination and Unfolded Protein Response in the Ocular Hypertensive Human Retina
Table 1
 
Proteins Linked to Protein Ubiquitination and Unfolded Protein Response in the Ocular Hypertensive Human Retina
Table 2
 
Proteins Linked to Cellular Redox Homeostasis and Oxidative Stress Response in the Ocular Hypertensive Human Retina
Table 2
 
Proteins Linked to Cellular Redox Homeostasis and Oxidative Stress Response in the Ocular Hypertensive Human Retina
Table 3
 
Proteins Linked to Cellular Energy Production and Mitochondrial Dysfunction in the Ocular Hypertensive Human Retina
Table 3
 
Proteins Linked to Cellular Energy Production and Mitochondrial Dysfunction in the Ocular Hypertensive Human Retina
Thus, ocular hypertensive retinas exhibited prominent downregulation of mitochondrial oxidative phosphorylation and increased antioxidant response. However, it is worth noting that despite the evidence suggesting a decrease in mitochondrial energy generation, as indicated in Figure 1, some ocular hypertensive samples also exhibited increased activity in glycolysis pathway, including over 2-fold increase in enolase (ENO2, Accession ID: P09104) and pyruvate kinase (PKM, Accession ID: P14618) expression in samples 1 and 3. 
As presented in Figures 2, 3, and 4, additional analyses were performed for validation of proteomics data, which included Western blot analysis for selected proteins, HSPA8 (a stress protein), SOD1 (an antioxidant enzyme), and five components of mitochondrial oxidative phosphorylation. Consistent with the proteomics data, Western blots indicated increased expression of HSPA8 (Fig. 2) and SOD1 (Fig. 3), but decreased expression of mitochondrial oxidative phosphorylation complexes (Fig. 4) in ocular hypertensive samples relative to normotensive controls. In addition, immunohistochemical analysis of HSPA8 (Fig. 2) and SOD1 (Fig. 3) supported protein localization in RGCs in the ocular hypertensive retina. 
Figure 2
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples validated increased expression of heat shock cognate protein 71 (HSPA8), a stress protein, in ocular hypertensive donor eyes (OHT-1 through OHT-6) relative to six age- and sex-matched normotensive healthy controls (C). Accompanying graph indicates the fold increase in beta-actin–normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls. Retinal immunolabeling with a specific antibody to HSPA8 (green) also indicated localization of this protein in NeuN+ neurons (red) in the retinal ganglion cell layer (RGCL). Blue corresponds to nuclear DAPI staining. Arrows indicate HSPA8-expressing neurons (yellow), likely corresponding to RGCs. Other retinal neurons in the inner nuclear layer (INL) or outer nuclear layer (ONL) of the ocular hypertensive retina exhibited weak immunolabeling for HSPA8. Presented images were obtained from sample pair 5 (scale bar: 100 μm).
Figure 2
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples validated increased expression of heat shock cognate protein 71 (HSPA8), a stress protein, in ocular hypertensive donor eyes (OHT-1 through OHT-6) relative to six age- and sex-matched normotensive healthy controls (C). Accompanying graph indicates the fold increase in beta-actin–normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls. Retinal immunolabeling with a specific antibody to HSPA8 (green) also indicated localization of this protein in NeuN+ neurons (red) in the retinal ganglion cell layer (RGCL). Blue corresponds to nuclear DAPI staining. Arrows indicate HSPA8-expressing neurons (yellow), likely corresponding to RGCs. Other retinal neurons in the inner nuclear layer (INL) or outer nuclear layer (ONL) of the ocular hypertensive retina exhibited weak immunolabeling for HSPA8. Presented images were obtained from sample pair 5 (scale bar: 100 μm).
Figure 3
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples validated increased expression of superoxide dismutase 1 (SOD1), an antioxidant enzyme, in ocular hypertensive donor eyes (OHT-1 through OHT-6) relative to six age- and sex-matched normotensive healthy controls (C). Accompanying graph indicates the fold increase in beta-actin-normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls. Retinal immunolabeling with a specific antibody to SOD1 (green) also indicated localization of this protein in NeuN+ neurons (red) in the retinal ganglion cell layer (RGCL). Blue corresponds to nuclear DAPI staining. Arrows indicate SOD1-expressing neurons (yellow), likely corresponding to RGCs. Other retinal neurons in the inner nuclear layer (INL) or outer nuclear layer (ONL) of the ocular hypertensive retina exhibited weak immunolabeling for SOD1. Presented images were obtained from sample pair 5 (scale bar: 100 μm).
Figure 3
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples validated increased expression of superoxide dismutase 1 (SOD1), an antioxidant enzyme, in ocular hypertensive donor eyes (OHT-1 through OHT-6) relative to six age- and sex-matched normotensive healthy controls (C). Accompanying graph indicates the fold increase in beta-actin-normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls. Retinal immunolabeling with a specific antibody to SOD1 (green) also indicated localization of this protein in NeuN+ neurons (red) in the retinal ganglion cell layer (RGCL). Blue corresponds to nuclear DAPI staining. Arrows indicate SOD1-expressing neurons (yellow), likely corresponding to RGCs. Other retinal neurons in the inner nuclear layer (INL) or outer nuclear layer (ONL) of the ocular hypertensive retina exhibited weak immunolabeling for SOD1. Presented images were obtained from sample pair 5 (scale bar: 100 μm).
Figure 4
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples validated downregulation of mitochondrial oxidative phosphorylation in ocular hypertensive donor eyes (OHT-1 through OHT-6) relative to six age- and sex-matched normotensive healthy controls (C). Immunoblotting used a cocktail of primary antibodies against complex I, NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8 (NDUFB8); complex II, succinate dehydrogenase complex subunit B (SDHB); complex III, ubiquinol-cytochrome c reductase core protein II (UQCRC2); complex IV, cytochrome c oxidase subunit II (COX2); and complex V, ATP synthase subunit alpha 1 (ATP5A1). Accompanying graph indicates the fold decrease in beta-actin-normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls.
Figure 4
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples validated downregulation of mitochondrial oxidative phosphorylation in ocular hypertensive donor eyes (OHT-1 through OHT-6) relative to six age- and sex-matched normotensive healthy controls (C). Immunoblotting used a cocktail of primary antibodies against complex I, NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8 (NDUFB8); complex II, succinate dehydrogenase complex subunit B (SDHB); complex III, ubiquinol-cytochrome c reductase core protein II (UQCRC2); complex IV, cytochrome c oxidase subunit II (COX2); and complex V, ATP synthase subunit alpha 1 (ATP5A1). Accompanying graph indicates the fold decrease in beta-actin-normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls.
Despite Molecular Risk Factors for Injury, No Molecular Alterations Supportive of an Ongoing Cell Death or Neuroinflammation Process Were Detectable in the Studied Samples of Human Retinas With Ocular Hypertension
In spite of molecular alterations supportive of mitochondrial dysfunction, endoplasmic reticulum stress, and adaptive/protective responses against tissue stress, no proteomic alterations consistent with an ongoing cell death process (such as increase in proapoptotic protein Bax, or proteolytic caspases or calpains as detected in the glaucomatous human retina21) were detectable through the analysis of ocular hypertensive retinal protein samples. Also noticeable in these samples were molecular alterations reflecting the modulatory responses against mitochondrial apoptosis pathway. As presented in Table 3, such molecular alterations included downregulation of voltage-dependent anion channels (VDAC) that are involved in mitochondrial permeability transition pore opening,27 and downregulation of apoptosis-inducing factor (AIF) that is involved in RGC apoptosis signaling in the glaucomatous human retina.21 
Table 4 lists additional proteins due to their potential relevance to glaucoma. These proteins include the marker proteins for two major cell types in the retina relevant to glaucoma, namely RGCs and astroglia. The RGC marker Thy-1 membrane glycoprotein was downregulated in two ocular hypertensive samples, but not significantly changed in other samples. The astroglial protein glial fibrillary acidic protein (GFAP) was downregulated in two ocular hypertensive samples but upregulated in the other four samples. 
Table 4
 
Glaucoma-Relevant Miscellaneous Proteins in the Ocular Hypertensive Human Retina
Table 4
 
Glaucoma-Relevant Miscellaneous Proteins in the Ocular Hypertensive Human Retina
As shown in Figure 1, besides increased expression of DNA repair enzymes, our proteomics datasets also included proteins linked to DNA methylation and transcriptional repression in the ocular hypertensive retina. Therefore, altered expression of related proteins is also listed in Table 4
No molecular alterations supportive of RGC death were detectable; however, as shown in Figure 1, some molecular alterations were associated with synaptic depression in the ocular hypertensive retina. As also presented in Table 4, these molecular alterations included over 2-fold downregulation of vesicular glutamate transporter, vGluT1, and metabotropic glutamate receptor, mGluR3, as well as downstream signaling. 
Also presented in Table 4 is tau protein implied in the pathology of Alzheimer's disease. No alteration (not over 2-fold) was detectable in the expression of tau protein in ocular hypertensive samples by proteome analysis; however, the signaling pathways that the Ingenuity Pathway Analysis associated with our datasets also pointed to amyloid processing toward tau hyperphosphorylation. To explore this association, Western blot analysis also used a specific antibody to p-tau, and immunoblots revealed increased p-tau in ocular hypertensive samples. In addition, tissue immunolabeling with the same p-tau antibody indicated prominent labeling in nerve fibers, as well as some labeling in RGC somas and dendrites in the ocular hypertensive retina (Fig. 5). 
Figure 5
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples using a phosphorylation site-specific antibody indicated increased expression of phosphorylated tau (p-tau), a microtubule protein associated with the pathology of Alzheimer's disease, in ocular hypertensive (OHT) donor eyes relative to six age- and sex-matched normotensive healthy controls (C). Accompanying graph indicates the fold increase in beta-actin–normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls. Retinal immunolabeling with the same antibody (green) also indicated prominent localization of this protein in the retinal nerve fiber layer. Presented images were obtained from sample pair 5 (scale bar: 100 μm). White box indicates the area of retinal nerve fibers shown in higher magnification. NeuN+ neurons (red) in the retinal ganglion cell layer (RGCL), inner nuclear layer (INL), and outer nuclear layer (ONL) of the ocular hypertensive retina also exhibited some immunolabeling for p-tau. Blue corresponds to nuclear DAPI staining.
Figure 5
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples using a phosphorylation site-specific antibody indicated increased expression of phosphorylated tau (p-tau), a microtubule protein associated with the pathology of Alzheimer's disease, in ocular hypertensive (OHT) donor eyes relative to six age- and sex-matched normotensive healthy controls (C). Accompanying graph indicates the fold increase in beta-actin–normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls. Retinal immunolabeling with the same antibody (green) also indicated prominent localization of this protein in the retinal nerve fiber layer. Presented images were obtained from sample pair 5 (scale bar: 100 μm). White box indicates the area of retinal nerve fibers shown in higher magnification. NeuN+ neurons (red) in the retinal ganglion cell layer (RGCL), inner nuclear layer (INL), and outer nuclear layer (ONL) of the ocular hypertensive retina also exhibited some immunolabeling for p-tau. Blue corresponds to nuclear DAPI staining.
Similar to the lack of cell death mediators, no inflammatory mediators were detectable in any of the studied retinal protein samples (such as increase in cytokines or chemokines, or nuclear factor-kappa B [NF-κB]-mediated transcriptional activation or signaling of inflammation as detected in the glaucomatous human retina20,21). The only detectable components of the immune system in these samples included some complement molecules and regulators, and the Ingenuity Pathway Analysis indicated association of the complement system with four out of six ocular hypertensive samples (samples 3–6). Therefore, Table 4 also presents complement components and regulatory molecules exhibiting altered expression in ocular hypertensive samples. There was over 2-fold upregulation of C3 in the two ocular hypertensive samples, while regulatory molecules (including C1q-binding protein and C1-inhibiting factor) exhibited downregulation. As a matter of interest, an inflammatory complement component, C4A, was detectable in sample 2, but its expression exhibited over 2-fold downregulation. 
Discussion
Proteomics analysis of human donor retinas provided new information about ocular hypertension–related molecular risk factors that may potentially distress the physiological equilibrium toward glaucoma development in human eyes. Besides presenting hypothesis-generating new information, findings of this study also provide a critical human validation for various aspects of recent experimental findings from animal models of glaucoma. 
Proteome analysis by LC-MS/MS and isotope labeling-based relative quantification of protein expression indicated prominent molecular activity in human donor retinas with ocular hypertension relative to ocular normotensive controls. Major molecular responses detected in the ocular hypertensive retina included various components of cell stress machinery and adaptive/protective responses for maintenance of cellular homeostasis. The cellular stress response characterized by upregulation of various stress-induced proteins is similar to proteomics data from human donor eyes with glaucoma.20,21 However, no proteomic alterations suggestive of an ongoing cell death process or neuroinflammation (both are evident in proteomics studies of human glaucoma1921,28) were detectable in ocular hypertensive retinas. Thus, the retinal proteome in ocular hypertensive eyes seems to reflect the intermediate between normal physiological conditions (as in nonglaucomatous healthy control donors) and glaucoma with ongoing neurodegeneration signaling (as in donors with glaucoma). 
Mitochondria play an important role in energy production through the multienzyme pathway of oxidative phosphorylation. We detected a prominent downregulation of many enzymes of oxidative phosphorylation in the ocular hypertensive retinas, which might signify a metabolic failure. Glucose is the main substrate for the brain's energy generation, and despite some controversies, its metabolism is mainly through oxidation in neurons and glycolysis in astrocytes.28,29 Therefore, downregulation of mitochondrial oxidative phosphorylation in the ocular hypertensive retina may more likely affect RGCs. It is also important to note that the increased expression of some glycolytic enzymes in ocular hypertensive retinas may suggest that glycolysis became enhanced to compensate for the weakened function of oxidative phosphorylation to maintain the ATP yield and meet the cellular energy demand in those retinas. Indeed, oxidative phosphorylation and glycolysis cooperate to maintain the cellular energy balance,30 and the retina has a similar capacity to switch between these two ATP-producing pathways.31 With respect to high energy demand of RGCs and their axons and high dependency of neuronal energy production on oxidative phosphorylation,28 a potential insufficiency in the energy supply could initiate defects in electrical conduction and axonal transport, thereby leading to neuron loss in ocular hypertensive eyes. Indeed, despite the lack of clinically detectable injury in our ocular hypertensive donor eyes, retinal proteomics/bioinformatics datasets were suggestive of weakening in synaptic transmission. We detected downregulation of glutamate transport and mGluR signaling in the ocular hypertensive human retina. Retinal vGluTs32 and mGluRs33 have been shown to be differentially regulated in ocular hypertensive DBA-2J mice. Based on the dynamic interplay between synaptic excitation and feedback inhibition, the downregulation of synaptic inhibition in some ocular hypertensive retinas might reflect a response to weakened synaptic activity for maintaining the visual function.34,35 This is interesting to further investigate, because patients with glaucoma exhibit early loss in retinal sensitivity,36,37 and early dysfunction of retinal synapses precedes structural damage in animal models of glaucoma.38,39 
In addition to declined ATP production, another pathogenic consequence of inhibited oxidative phosphorylation is increase in free radical generation leading to oxidative stress. Electrons leaked from complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), and complex III (ubiquinol-cytochrome c reductase) are taken up by oxygen to form reactive oxygen species. Increased oxidant production further impairs mitochondrial function, thereby creating a vicious cycle. Previous evidence has implicated mitochondria as an important source of oxidative stress in glaucoma.40,41 Our data from the ocular hypertensive human retina suggest a similar contribution of mitochondria in ocular hypertension-induced oxidative stress. 
Besides the upregulated expression of stress-responsive heat shock proteins, ocular hypertension–related tissue stress in the human retina was evident by a prominent antioxidant response reflecting the oxidative stress and endogenous efforts to reduce oxidative stress–related damage. The knowledge-based analysis by the Ingenuity Pathway Analysis associated the ocular hypertension–related oxidative stress response with NRF2. The NRF2 is a key transcription factor that promotes antioxidant enzymes and detoxifying enzymes42 and plays a critical role in cellular defense against oxidative stress, including in RGCs.43 The increased expression of antioxidant enzymes in the ocular hypertensive human retina is similar to previous proteomics data from human glaucoma21 and experimental glaucoma.23 However, different from ocular hypertensive retina, the free radical increase in glaucomatous samples exceeds the intrinsic antioxidant capacity, thereby resulting in retinal protein oxidation.22,44,45 Oxidative damage to proteins, lipids, and DNA leads to structural and functional alterations, and possibly promotes neuronal cell death, glial dysfunction, and inflammation.7,8 The upregulated antioxidant response that we detected in the ocular hypertensive human retina may therefore indicate oxidative stress as a molecular risk factor for glaucoma development. This is because if antioxidant defense cannot overcome the oxidant generation, then neurons become vulnerable to oxidative stress-related damage as evident in glaucoma. 
Thus, the consistency in downregulation of mitochondrial phosphorylation proteins among six ocular hypertensive samples versus six controls, along with a prominent oxidative stress response in the same samples with ocular hypertension, supports ocular hypertension–induced mitochondrial dysfunction. Mitochondria also play a central role in apoptosis cascade; the mitochondrial membrane potential that drives oxidative phosphorylation decreases during apoptosis, and maintaining the cellular ATP level is an important determinant in preventing the apoptotic cell death. However, the studied human retinas did not exhibit the activation of cell death signaling through mitochondria-mediated or death receptor-mediated caspase cascades, but instead downregulation of molecules involved in initiating the mitochondrial apoptosis pathway, including VDAC or AIF. With respect to important roles of mitochondria in glaucoma9,40,41 and evidence of deteriorating mitochondria in ocular hypertension–induced animal models of glaucoma,46,47 our observations in the ocular hypertensive human retina motivate further functional analysis to determine whether mitochondrial energy insufficiency and generated oxidative stress are early events promoting neurodegeneration in human glaucoma, and whether mitochondrial failure may serve as a treatment target. 
Another similarity of the ocular hypertensive retina to glaucomatous retina was increased expression of endoplasmic reticulum-resident stress-regulated chaperones that catalyze protein folding and function as sensors detecting unfolded protein response.48 Upregulation of unfolded protein response (for protein repair/removal) and ubiquitination (required for targeted degradation of proteins that are not properly folded within the endoplasmic reticulum) may be indicators for endoplasmic reticulum stress in the ocular hypertensive retina. Although such responses represent endogenous adaptive efforts to preserve cell viability and function, persistence of disturbances in endoplasmic reticulum homeostasis and resultant redox changes may also contribute to mitochondria-generated oxidative stress, thereby further increasing the neuronal susceptibility to glaucomatous injury.49,50 Endoplasmic reticulum stress and impairment or overburdening of the ubiquitin proteasome pathway51 have been associated with pathogenic mechanisms of glaucoma in in vitro52,53 and in vivo models.5456 
Thus, ocular hypertensive human retina with no manifest neuronal injury presented various components of tissue stress and intrinsic adaptive/protective responses that are also evident in the glaucomatous human retina. Also in parallel to previous observations in glaucoma, many signaling pathways, mainly linked to regulation of cell growth and cell protection, were associated with our ocular hypertensive human datasets. These pathways included the mTOR signaling regulating autophagy,23,57 Rho family GTPases,58 ephrin receptor signaling,59 integrin signaling,60 14-3-3-mediated signaling,61 CREB,62 ERK/MAPK signaling,63,64 PI3K/Akt signaling,65 calcium signaling,66,67 neurotrophin/TRK signaling,68,69 JAK/STAT signaling,21 and endothelin-1 signaling.70 It is important to emphasize that since the studied ocular hypertensive human retinas did not exhibit the activation of cell death signaling, the cell protective responses detected in these samples were more likely to be sufficient for protecting neurons against ocular hypertension–induced glaucomatous injury. Alternatively, our samples, including punches and central-to-peripheral wedges of the retina, might not well reflect the regional pattern of neuron loss in early glaucoma. However, additional observations, including increased DNA methylation and transcriptional repression,21 and downregulated expression of Thy-1,7173 also seem analogous to early molecular alterations detected prior to neuronal damage in ocular hypertensive animal models. In addition, our proteomics data from the ocular hypertensive human retina are somehow parallel to gene array data from the anterior segment tissues of patients with pseudoexfoliation syndrome (without glaucoma), which also reflect molecular alterations prior to clinically manifest disease (in this case, a secondary type of glaucoma).74 The gene array study of anterior segment tissues with pseudoexfoliation has indicated molecular alterations in cytoprotective mechanisms comparable to that we detected in the ocular hypertensive human retina. 
Similar to the lack of cell death signaling, the stage of ocular hypertension–induced molecular responses in our samples did not present alterations implying neuroinflammation. For example, proteome analysis of the ocular hypertensive human retina did not detect any increase in cytokine or chemokine production, or increase in molecules involved in inflammatory signaling through tumor necrosis factor or toll-like receptors, NF-κB activation, or inflammasome assembly, all of which are evident in human glaucoma.20,21 Despite the lack of glia-driven neuroinflammatory activity, we detected upregulation of GFAP in four samples and downregulation in two samples. Upregulation of GFAP expression has conventionally been viewed as a hallmark of astroglial reactivity to glaucomatous tissue stress.63,75,76 The inconsistency detected in GFAP expression among ocular hypertensive samples may be explained by spatial distribution of astroglial reactivity (and spatial pattern of upregulated or downregulated expression of GFAP)77 and its dependence on the stage of ocular hypertension–induced responses78 in experimental models. The absence of detectable neuroinflammatory responses may bring up the suggestion that the retinal inflammatory activity is merely secondary to neuronal injury, which was not evident in our ocular hypertensive human donor eyes. Alternatively, our ocular hypertensive samples might have been at the stages in which neurodegenerative inflammation had not yet started. For example, adaptive/protective responses detected in the ocular hypertensive retina, including the prominent antioxidant response (which may affect redox-sensitive transcriptional regulation of immune mediators by NF-κB79), might have been sufficient to repress proinflammatory activation at early stages. It also remains unclear whether the lack of neuroinflammation was actually protective in those ocular hypertensive donors who had not developed glaucoma. These possibilities stimulate further studies of animal models closely mimicking the conditions in human glaucoma, as well as additional studies of human donor tissues to better understand and treat the inflammatory component of human glaucoma. 
Although no proteomic alterations suggestive of inflammation were detectable, there was a trend toward complement activation in the ocular hypertensive retina as characterized by increased expression of C3 and decreased expression of complement regulators. This observation that is similar to the glaucomatous human retina22 may be pertinent to the synaptic depression noted in ocular hypertensive retinas because complement activation has been proposed to be involved in early loss of RGC synapses in experimental glaucoma.80 It would therefore be interesting to further pursue whether complement-mediated tissue cleaning is involved in elimination of dysfunctional RGC synapses in the ocular hypertensive human retina, and whether complement-mediated collateral injury to RGCs precedes human glaucoma. 
Another interesting finding in ocular hypertensive retinas was prominently increased p-tau. The slow and progressive neurodegeneration characteristic of Alzheimer's disease involves the formation of amyloid plaques and neurofibrillary tangles, composed of hyperphosphorylated tau.81 While normal tau promotes assembly and stabilization of microtubules, abnormally phosphorylated tau disrupts these cellular structures. Various factors, including oxidative stress, may lead to a vicious cycle of cellular events that may cause increased phosphorylation and polymerization of tau during the lifetime.82 Disruption of microtubules may impair the axoplasmic flow, thereby leading to slowly progressive retrograde degeneration. Indeed, tau inclusions in RGCs affect the axonal viability,83 and besides its retinal manifestation,84 Alzheimer's disease may share pathogenic mechanisms with glaucoma.85,86 Amyloid precursor protein has been shown to be abnormally processed, and neurotoxic amyloid-β species have been found upregulated in the retina of experimental glaucoma models.8789 Tau phosphorylation has also been detected in the glaucomatous human retina.90 Our similar observation in the ocular hypertensive human retina with no manifest glaucoma may suggest the role of p-tau in disease development, which is also worth pursuing in further research. 
It should be clarified that the findings of this study provide unique information but also challenges intrinsic to human donor tissues. To minimize any vulnerability related to individual differences in protein expression, our samples (six ocular hypertensive samples and six normotensive controls) were carefully paired by matching for donor age and sex (as well as the history of systemic or eye disease and the postmortem period till sample collection). As seen in the proteomics data tables and Western blot images (both presented side by side for each of the six sample pairs), despite individual variability in protein expression among samples with different age (most prominent in HSPA8 and p-tau immunoblots), the overall protein expression trend was consistent among the six sample pairs (cellular stress and adaptive/protective responses in ocular hypertensive samples versus controls). In addition, we preferred not to include the macular region (which is rich in RGCs) in this study due to common aging-related alterations in this region of the human retina, which otherwise could interfere with hypertension–related alterations. Although experimental studies of animal models allow proteomics analysis of cell type–enriched samples to reduce sample complexity,14,15 human donor tissues with limited availability are not suitable for this type of analysis. However, besides retinal punches subjected to proteomics and Western blot analyses, the studied samples in this study included central-to-peripheral wedges of the retina from the same donors for immunohistochemical analysis of selected proteins. This analysis indicated prominent localization of the upregulated proteins, including HSPA8, SOD1, and p-tau, in RGC or nerve fiber layers of the ocular hypertensive retina. These observations support that our data well reflect the molecules/pathways affected early by ocular hypertension (typically in the inner retina), rather than secondary responses in other retinal layers. Although our human donors had well-documented clinical records, due to the retrospective nature of donor data collection (clinical data were recorded at multiple clinical visits over years and retina samples were collected post mortem) and the changeability of intraocular pressure/systemic health conditions/medications during a period of up to over 10 years, we considered that determining any correlation of the protein expression data with such clinical parameters would not be precisely informative. Nevertheless, molecular alterations detected in this proteomics study of ocular hypertensive human donor retinas seem correlative to clinical observations in patients with ocular hypertension. Of particular interest, prediction models for glaucoma development in ocular hypertensive individuals indicate “baseline age” as the major risk factor.91 This clinical observation may be related to augmentation of hypertension–related oxidative stress (detected in this study) by aging-related oxidative stress (a common component of aging-related pathogenic processes) in increasing the risk for disease development. 
In conclusion, the presented proteomics data provide new clues about ocular hypertension–related molecular alterations in the human retina (Fig. 6). Despite an intrinsic stress response, there was no detectable activation of cell death pathways or neuroinflammation. The molecular information about ocular hypertension–related early alterations in the human retina, as opposed to alterations detected in eyes with clinically manifest glaucomatous damage, supports that proteome alterations determine the “threshold” for vulnerability of neurons to injury (Fig. 7). Individual factors (genetic/epigenetic, local/systemic) likely affect such a cellular stressor threshold to tolerate ocular hypertension–induced tissue stress or convert to neurodegenerative injury when endogenous adaptive/protective responses are overwhelmed or impaired. Findings of this study encourage further research to better understand ocular hypertension–related molecular risk factors for glaucoma conversion and to thereby develop new treatment strategies for neuroprotection. 
Figure 6
 
Ocular hypertension–related molecular alterations in the human retina included mitochondrial dysfunction leading to energy failure and increased production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress, which resulted in antioxidant response and unfolded protein response (UPR). Other molecular responses to ocular hypertension may include DNA methylation/transcriptional repression, glial activation, complement upregulation (and synapse loss), and abnormal amyloid processing.
Figure 6
 
Ocular hypertension–related molecular alterations in the human retina included mitochondrial dysfunction leading to energy failure and increased production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress, which resulted in antioxidant response and unfolded protein response (UPR). Other molecular responses to ocular hypertension may include DNA methylation/transcriptional repression, glial activation, complement upregulation (and synapse loss), and abnormal amyloid processing.
Figure 7
 
Ocular hypertension–related molecular alterations in the human retina as opposed to alterations detected in eyes with clinically manifest glaucoma support that proteome alterations determine the “threshold” for vulnerability of neurons to injury. This threshold, depending on individual factors, appears to be critical to tolerating the ocular hypertension–induced tissue stress or developing glaucoma if endogenous adaptive/protective responses are overwhelmed.
Figure 7
 
Ocular hypertension–related molecular alterations in the human retina as opposed to alterations detected in eyes with clinically manifest glaucoma support that proteome alterations determine the “threshold” for vulnerability of neurons to injury. This threshold, depending on individual factors, appears to be critical to tolerating the ocular hypertension–induced tissue stress or developing glaucoma if endogenous adaptive/protective responses are overwhelmed.
Acknowledgments
Supported in part by the National Eye Institute, Bethesda, Maryland, United States (1R21EY024105); Glaucoma Research Foundation, San Francisco, California, United States; and Research to Prevent Blindness, Inc., New York, New York, United States, providing an unrestricted grant to the Columbia University Department of Ophthalmology. GT is the recipient of the Homer McK. Rees Scholarship in Glaucoma Research and an awardee of the Peacock Trusts. 
Disclosure: X. Yang, None; G. Hondur, None; M. Li, None; J. Cai, None; J.B. Klein, None; M.H. Kuehn, None; G. Tezel, None 
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Figure 1
 
Bioinformatics analysis of proteomics datasets. Retinal protein samples from ocular hypertensive human retinas and normotensive controls were analyzed by two-dimensional LC-MS/MS using oxygen isotope labeling for relative quantification of protein expression. Quantitative proteomics datasets were then searched for functional patterns by the Ingenuity Pathway Analysis. This analysis linked the ocular hypertension–related retinal proteomic alterations to various canonical pathways from the Ingenuity knowledge library, including those shown in this graph. The bars display the significance of these associations (the −log of P value calculated by Fisher's exact test right-tailed), and the orange squares connected by a thin line represent the ratio of the number of proteins in our dataset of a given pathway to the total number of proteins in this canonical pathway.
Figure 1
 
Bioinformatics analysis of proteomics datasets. Retinal protein samples from ocular hypertensive human retinas and normotensive controls were analyzed by two-dimensional LC-MS/MS using oxygen isotope labeling for relative quantification of protein expression. Quantitative proteomics datasets were then searched for functional patterns by the Ingenuity Pathway Analysis. This analysis linked the ocular hypertension–related retinal proteomic alterations to various canonical pathways from the Ingenuity knowledge library, including those shown in this graph. The bars display the significance of these associations (the −log of P value calculated by Fisher's exact test right-tailed), and the orange squares connected by a thin line represent the ratio of the number of proteins in our dataset of a given pathway to the total number of proteins in this canonical pathway.
Figure 2
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples validated increased expression of heat shock cognate protein 71 (HSPA8), a stress protein, in ocular hypertensive donor eyes (OHT-1 through OHT-6) relative to six age- and sex-matched normotensive healthy controls (C). Accompanying graph indicates the fold increase in beta-actin–normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls. Retinal immunolabeling with a specific antibody to HSPA8 (green) also indicated localization of this protein in NeuN+ neurons (red) in the retinal ganglion cell layer (RGCL). Blue corresponds to nuclear DAPI staining. Arrows indicate HSPA8-expressing neurons (yellow), likely corresponding to RGCs. Other retinal neurons in the inner nuclear layer (INL) or outer nuclear layer (ONL) of the ocular hypertensive retina exhibited weak immunolabeling for HSPA8. Presented images were obtained from sample pair 5 (scale bar: 100 μm).
Figure 2
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples validated increased expression of heat shock cognate protein 71 (HSPA8), a stress protein, in ocular hypertensive donor eyes (OHT-1 through OHT-6) relative to six age- and sex-matched normotensive healthy controls (C). Accompanying graph indicates the fold increase in beta-actin–normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls. Retinal immunolabeling with a specific antibody to HSPA8 (green) also indicated localization of this protein in NeuN+ neurons (red) in the retinal ganglion cell layer (RGCL). Blue corresponds to nuclear DAPI staining. Arrows indicate HSPA8-expressing neurons (yellow), likely corresponding to RGCs. Other retinal neurons in the inner nuclear layer (INL) or outer nuclear layer (ONL) of the ocular hypertensive retina exhibited weak immunolabeling for HSPA8. Presented images were obtained from sample pair 5 (scale bar: 100 μm).
Figure 3
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples validated increased expression of superoxide dismutase 1 (SOD1), an antioxidant enzyme, in ocular hypertensive donor eyes (OHT-1 through OHT-6) relative to six age- and sex-matched normotensive healthy controls (C). Accompanying graph indicates the fold increase in beta-actin-normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls. Retinal immunolabeling with a specific antibody to SOD1 (green) also indicated localization of this protein in NeuN+ neurons (red) in the retinal ganglion cell layer (RGCL). Blue corresponds to nuclear DAPI staining. Arrows indicate SOD1-expressing neurons (yellow), likely corresponding to RGCs. Other retinal neurons in the inner nuclear layer (INL) or outer nuclear layer (ONL) of the ocular hypertensive retina exhibited weak immunolabeling for SOD1. Presented images were obtained from sample pair 5 (scale bar: 100 μm).
Figure 3
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples validated increased expression of superoxide dismutase 1 (SOD1), an antioxidant enzyme, in ocular hypertensive donor eyes (OHT-1 through OHT-6) relative to six age- and sex-matched normotensive healthy controls (C). Accompanying graph indicates the fold increase in beta-actin-normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls. Retinal immunolabeling with a specific antibody to SOD1 (green) also indicated localization of this protein in NeuN+ neurons (red) in the retinal ganglion cell layer (RGCL). Blue corresponds to nuclear DAPI staining. Arrows indicate SOD1-expressing neurons (yellow), likely corresponding to RGCs. Other retinal neurons in the inner nuclear layer (INL) or outer nuclear layer (ONL) of the ocular hypertensive retina exhibited weak immunolabeling for SOD1. Presented images were obtained from sample pair 5 (scale bar: 100 μm).
Figure 4
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples validated downregulation of mitochondrial oxidative phosphorylation in ocular hypertensive donor eyes (OHT-1 through OHT-6) relative to six age- and sex-matched normotensive healthy controls (C). Immunoblotting used a cocktail of primary antibodies against complex I, NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8 (NDUFB8); complex II, succinate dehydrogenase complex subunit B (SDHB); complex III, ubiquinol-cytochrome c reductase core protein II (UQCRC2); complex IV, cytochrome c oxidase subunit II (COX2); and complex V, ATP synthase subunit alpha 1 (ATP5A1). Accompanying graph indicates the fold decrease in beta-actin-normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls.
Figure 4
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples validated downregulation of mitochondrial oxidative phosphorylation in ocular hypertensive donor eyes (OHT-1 through OHT-6) relative to six age- and sex-matched normotensive healthy controls (C). Immunoblotting used a cocktail of primary antibodies against complex I, NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8 (NDUFB8); complex II, succinate dehydrogenase complex subunit B (SDHB); complex III, ubiquinol-cytochrome c reductase core protein II (UQCRC2); complex IV, cytochrome c oxidase subunit II (COX2); and complex V, ATP synthase subunit alpha 1 (ATP5A1). Accompanying graph indicates the fold decrease in beta-actin-normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls.
Figure 5
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples using a phosphorylation site-specific antibody indicated increased expression of phosphorylated tau (p-tau), a microtubule protein associated with the pathology of Alzheimer's disease, in ocular hypertensive (OHT) donor eyes relative to six age- and sex-matched normotensive healthy controls (C). Accompanying graph indicates the fold increase in beta-actin–normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls. Retinal immunolabeling with the same antibody (green) also indicated prominent localization of this protein in the retinal nerve fiber layer. Presented images were obtained from sample pair 5 (scale bar: 100 μm). White box indicates the area of retinal nerve fibers shown in higher magnification. NeuN+ neurons (red) in the retinal ganglion cell layer (RGCL), inner nuclear layer (INL), and outer nuclear layer (ONL) of the ocular hypertensive retina also exhibited some immunolabeling for p-tau. Blue corresponds to nuclear DAPI staining.
Figure 5
 
Altered protein expression in the ocular hypertensive human retina. Western blot analysis of retinal protein samples using a phosphorylation site-specific antibody indicated increased expression of phosphorylated tau (p-tau), a microtubule protein associated with the pathology of Alzheimer's disease, in ocular hypertensive (OHT) donor eyes relative to six age- and sex-matched normotensive healthy controls (C). Accompanying graph indicates the fold increase in beta-actin–normalized average intensity values obtained from ocular hypertensive samples relative to ocular normotensive controls. Retinal immunolabeling with the same antibody (green) also indicated prominent localization of this protein in the retinal nerve fiber layer. Presented images were obtained from sample pair 5 (scale bar: 100 μm). White box indicates the area of retinal nerve fibers shown in higher magnification. NeuN+ neurons (red) in the retinal ganglion cell layer (RGCL), inner nuclear layer (INL), and outer nuclear layer (ONL) of the ocular hypertensive retina also exhibited some immunolabeling for p-tau. Blue corresponds to nuclear DAPI staining.
Figure 6
 
Ocular hypertension–related molecular alterations in the human retina included mitochondrial dysfunction leading to energy failure and increased production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress, which resulted in antioxidant response and unfolded protein response (UPR). Other molecular responses to ocular hypertension may include DNA methylation/transcriptional repression, glial activation, complement upregulation (and synapse loss), and abnormal amyloid processing.
Figure 6
 
Ocular hypertension–related molecular alterations in the human retina included mitochondrial dysfunction leading to energy failure and increased production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress, which resulted in antioxidant response and unfolded protein response (UPR). Other molecular responses to ocular hypertension may include DNA methylation/transcriptional repression, glial activation, complement upregulation (and synapse loss), and abnormal amyloid processing.
Figure 7
 
Ocular hypertension–related molecular alterations in the human retina as opposed to alterations detected in eyes with clinically manifest glaucoma support that proteome alterations determine the “threshold” for vulnerability of neurons to injury. This threshold, depending on individual factors, appears to be critical to tolerating the ocular hypertension–induced tissue stress or developing glaucoma if endogenous adaptive/protective responses are overwhelmed.
Figure 7
 
Ocular hypertension–related molecular alterations in the human retina as opposed to alterations detected in eyes with clinically manifest glaucoma support that proteome alterations determine the “threshold” for vulnerability of neurons to injury. This threshold, depending on individual factors, appears to be critical to tolerating the ocular hypertension–induced tissue stress or developing glaucoma if endogenous adaptive/protective responses are overwhelmed.
Table 1
 
Proteins Linked to Protein Ubiquitination and Unfolded Protein Response in the Ocular Hypertensive Human Retina
Table 1
 
Proteins Linked to Protein Ubiquitination and Unfolded Protein Response in the Ocular Hypertensive Human Retina
Table 2
 
Proteins Linked to Cellular Redox Homeostasis and Oxidative Stress Response in the Ocular Hypertensive Human Retina
Table 2
 
Proteins Linked to Cellular Redox Homeostasis and Oxidative Stress Response in the Ocular Hypertensive Human Retina
Table 3
 
Proteins Linked to Cellular Energy Production and Mitochondrial Dysfunction in the Ocular Hypertensive Human Retina
Table 3
 
Proteins Linked to Cellular Energy Production and Mitochondrial Dysfunction in the Ocular Hypertensive Human Retina
Table 4
 
Glaucoma-Relevant Miscellaneous Proteins in the Ocular Hypertensive Human Retina
Table 4
 
Glaucoma-Relevant Miscellaneous Proteins in the Ocular Hypertensive Human Retina
Supplement 1
Supplement 2
Supplement 3
Supplement 4
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