Proteomic identification of oxidatively modified retinal proteins in a chronic pressure-induced rat model of glaucoma

Abstract

Purpose: Based on the evidence of an amplified production of reactive oxygen species (ROS) during glaucomatous neurodegeneration, proteomic analysis was performed to determine oxidative modification of retinal proteins after experimental elevation of intraocular pressure (IOP).

Methods: IOP elevation was induced in rats by hypertonic saline injections into episcleral veins. Protein expression was determined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of retinal protein lysates obtained from eyes matched for the cumulative IOP exposure and axon loss. To determine protein oxidation levels, protein carbonyls were detected through 2D-oxyblot analysis of 2,4-dinitrophenylhydrazine (DNPH)-treated samples using an anti-DNP antibody. For identification of oxidized proteins, peptide masses were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS). In addition to use of different engines in a bioinformatic database search and performance of peptide sequencing and 2D-Western blot analysis for confirmation of the identified proteins, immunohistochemistry was used for further validation of the proteomic findings.

Results: Comparison of 2D-oxyblots with Coomassie Blue-stained 2D-gels revealed that approximately 60 protein spots obtained with retinal protein lysates from ocular hypertensive eyes (of >400 spots) exhibited protein carbonyl immunoreactivity, which reflects oxidatively modified proteins. There was a significant increase in anti-carbonyl reactivity in individual protein spots obtained with retinal protein lysates from ocular hypertensive eyes compared with the control (P < 0.01). The identified proteins through peptide mass fingerprinting and peptide sequencing included glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme; HSP72, a stress protein; and glutamine synthetase, an excitotoxicity-related protein. Immunolabeling of retina sections with specific antibodies demonstrated cellular localization of these proteins as well as retinal distribution of the increased protein carbonyl immunoreactivity in ocular hypertensive eyes.

Conclusions: The findings of this in vivo study provide novel evidence for oxidative modification of many retinal proteins in ocular hypertensive eyes and identify three specific targets of retinal protein oxidation in these eyes, thereby supporting the association of oxidative damage with neurodegeneration in glaucoma. By using a proteomic approach, this study also exemplifies that proteomics provide a very promising way to elucidate pathogenic mechanisms in glaucoma at the protein level.

Figure 1.

Experimental rat model of chronic pressure-induced glaucoma. (A, B) The course of IOP elevation and axon loss, respectively, during an experimental period of up to 12 weeks after hypertonic saline injection into the limbal veins in rats. Data are presented as the mean ± SD. The percentage axon loss was determined by comparing the estimated total number of optic nerve axons in the ocular hypertensive eye relative to the control fellow eye. The degree of cumulative IOP exposure was estimated by first integrating IOP over time in the ocular hypertensive eye, then subtracting the IOP-time integral from that in the normotensive fellow eye (mm Hg-days). (C) The association of axon loss with cumulative IOP exposure.

Figure 2.

2D-PAGE of retinal protein lysates. Coomassie Blue-stained gels and oxyblots were obtained by 2D-PAGE of the equally loaded retinal protein samples from control or ocular hypertensive rat eyes. Protein carbonyl immunoreactivity detected on 2D-oxyblots, which reflects oxidatively modified proteins, occurred to a great extent in the retina of ocular hypertensive eyes. Arrows: protein spots identified through peptide mass fingerprinting and peptide sequencing as presented in Figures 4 and 5. PI, isoelectric point.

Figure 3.

2D-gels stained with Sypro-Ruby (Bio-Rad). Comparison between stained gel images of the same sample, DNPH-untreated (top) and DNPH-treated (bottom) showed no major differences in protein position.

Figure 4.

Peptide mass fingerprinting. The protein spots shown by arrows in Figure 2 were identified by using mass spectrometry and bioinformatics. The identified proteins (Fig. 2, numbers 1, 2, and 3) included a glycolytic enzyme, GAPDH; a stress protein, HSP72; and an excitotoxicity-related protein, glutamine synthetase, respectively. Left: mass spectra for these spots. Spectral masses (in mass per charge unit, M/z) obtained by MALDI-TOF/MS were analyzed by using bioinformatics through the Mascot and Profound search engines. Right: the probability-based Mowse scores obtained using the Mascot search engine. Among predicted proteins with differential Mowse scores shown as multiple bars on the x-axis, only proteins with Mowse scores greater than 71 (outside the shaded area) were considered significant, which were 113, 254, and 131 (P < 0.05) for GAPDH, HSP72, and glutamine synthetase, respectively. The second significant bar indicating a Mowse score of 80 for the sample corresponding to HSP72 was HSP70, the constitutive form of HSP72. The Profound search results were consistent with the Mascot results, and z-scores for all three proteins were 2.43 (P < 0.001). Asterisks indicate trypsin autolysis peaks.

Figure 5.

Peptide sequencing. The protein spots shown by arrows in Figure 2 were also analyzed by using liquid chromatography-tandem mass spectrometry (LC/MS/MS). This confirmed that the peptide sequences match with GAPDH, HSP72, and glutamine synthetase. LC/MS/MS spectra for selected peptides and their sequences are shown.

Figure 6.

Immunohistochemical analysis of the identified proteins. Retina sections obtained from control and ocular hypertensive rat eyes were subjected to immunofluorescence labeling using specific antibodies. The merged images presented in (A) and (B) show immunofluorescence labeling of the retina in control (A) and ocular hypertensive (B) eyes for GAPDH as green, and the nuclear DAPI labeling as blue. Retinal GAPDH immunolabeling included all cell types, as expected, and the intensity of GAPDH immunolabeling was similar in control and ocular hypertensive eyes. However, it is notable that many RGCs identified based on morphologic assessment (arrow-heads) exhibited prominent nuclear immunolabeling for GAPDH in ocular hypertensive eyes. (C) HSP72 immunolabeling of a control retina (green) was predominant in the inner retinal layers. (D) A similar pattern of HSP72 immunolabeling in the retina of an ocular hypertensive eye (green) under higher magnification. The merged image also shows GFAP immunolabeling. HSP72 immunolabeling was positive in both GFAP-positive astrocytes (yellow) and GFAP-negative neurons (green). The GFAP-negative neurons in the inner retina are most likely RGCs (arrows). Retinal immunolabeling for glutamine synthetase in the control (E) and ocular hypertensive (F) eyes, which corresponds to Müller cell bodies located in the inner nuclear layer and their processes in the inner and outer limiting membranes. No difference was detectable between the glutamine synthetase immunolabeling of the retina in control and ocular hypertensive eyes. gc, ganglion cell layer; in, inner nuclear layer; on, outer nuclear layer. Scale bar: (A, B, D) 50 μm; (C, E, F) 100 μm.

Figure 7.

Immunohistochemical analysis of protein carbonyl immunoreactivity. Retina sections obtained from control and ocular hypertensive rat eyes were subjected to immunofluorescence labeling for protein carbonyls. (A, B) Immunofluorescence labeling of DNPH-treated retinal sections with a specific anti-DNP antibody in control and ocular hypertensive eyes, respectively. The increased retinal protein carbonyl immunoreactivity in ocular hypertensive eyes compared with the controls was predominant in the inner retinal layers. (C) Merged image presented in (B) with another image (not shown) of the same region demonstrating brn-3 immunolabeling. Localization of anti-carbonyl reactivity to brn-3-positive RGCs (yellow) indicates that RGC proteins are among the retinal proteins exhibiting increased susceptibility to oxidative modification in ocular hypertensive eyes. Brn-3-negative cells exhibiting protein carbonyl immunoreactivity in the inner nuclear layer are likely the Müller cells. gc, ganglion cell; in, inner nuclear; on, outer nuclear layers. Scale bar, 100 μm.