Gene and protein expression pilot profiling and biomarkers in an experimental mouse model of hypertensive glaucoma

Abstract

Glaucoma is a group of genetically heterogeneous neurodegenerative disorders causing the degeneration of the ganglion neurons of the retina. Increased intraocular pressure (IOP) is a hallmark risk factor promoting the death of ganglion neurons of the retina in glaucoma. Yet, the molecular processes underlying the degeneration of these neurons by increased IOP are not understood. To gain insight into the early molecular events and discover biomarkers induced by IOP, we performed gene and protein expression profiling to compare retinas of eyes with and without high IOP in a rodent model of experimental glaucoma. This pilot study found that the IOP-mediated changes in the transcription levels of a restricted set of genes implicated in peroxisomal and mitochondrial function, modulation of neuron survival and inflammatory processes, were also accompanied by changes in the levels of proteins encoded by the same genes. With the exception of the inflammatory markers, serum amyloid-A1 (SAA1) and serum amyloid-A2 (SAA2), the IOP-induced changes in protein expression were restricted to ganglion neurons of the retina and they were detected also in the vitreous, thus suggesting an early IOP-mediated loss of ganglion cell integrity. Interestingly, SAA1 and SAA2 were induced in retinal microglia cells, whereas they were reduced in sera of IOP-responsive mice. Hence, this study defines novel IOP-induced molecular processes, biomarkers and sources thereof, and it further validates the extension of the analyses herein reported to other genes modulated by IOP.

Figure 1

Induction of high intraocular pressure (IOP) in a mouse model of experimental glaucoma without neuronal cell loss. A. IOPs begin to increase in injected eyes as compared with the control eyes at week 4 and sustained elevated pressures continue through week 7. Week 0 represents the baseline intraocular pressures just before the hypertonic saline injections were performed. Initially, there were similar pressures in both the injected and control eyes during weeks 0–3. IOPs reached maximal elevation as well as statistical significance at week 5 and they remained elevated throughout the duration of the study (week 7). Data are means of three independent experiments ± s.d. B. No significant neuronal cell loss of the ganglion cell layer of the retina was present between injected eyes at week-7 post-injection (time of sacrifice) and non-injected eyes. Cell loss was measured by comparing Hoechst-positive nuclei of cells of the ganglion cell layer between retinas of non-injected and injected eyes. Data presented as means of sample nuclei counts from all four quadrants of three retinas ± standard deviation. C. TUNEL staining (nucleosomal DNA fragmentation) of retinal sections of eyecups showed no apoptotic cell death in the ganglion cell layer (arrows) or any other nuclear layers of the retina between non-injected (Ctrl; left image) and injected (Inj; right image) eyes. Retinal sections were counterstained with DAPI (blue). Scale bar: 400 μm

Figure 2

Overall changes of gene expression by elevated IOP. Hierarchical clustering (heat plot) of 71 retinal genes whose modulation of expression by IOP was shared by volcano and pairwise analyses of untreated (control) left (L) and contralateral treated (injected) right (R) eyes (n=3). Branch lengths of dendrogram on the left depict the relatedness of expression patterns. Genes with higher expression are depicted in red; genes with lower expression are shown in green.

Figure 3

Elevated IOP promotes altered expression of proteins in the retina encoded by the genes with IOP-induced change of transcription (as noted in Table 1). A. Western blot analysis of extracts of retinas with (injected; Inj) and without (control, Ctrl) exposure to elevated IOP. When compared to control retinas, Bcl2 levels were decreased in retinas of contralateral injected eyes, whereas the levels of catalase (Cat) and XIAP were increased. Tubulin serves as a loading control. Retinas were exposed to 3-weeks of elevated IOP during the 7-week post-injection treatment as those shown in Fig 1A. B. Fold change of protein expression in retinas of injected eyes compared to control eyes (n=3). Quantitation of protein levels ascertained by Western blot showed an almost 50% reduction in Bcl2 expression, a 4.5-fold increase in catalase expression, and a 3-fold increase in XIAP expression in the eyes with elevated intraocular pressure as compared with the controls. C. Western blot of retinas of injected eyes of non-responders, which were refractory to the single injection treatment (without IOP elevation), and control (non-injected) eyes. The protein expression levels of Bcl2, Cat, and XIAP are comparable in the retinas of both the injected and control eyes. Tubulin served as a loading control. Retinas were collected 7-weeks after the injection treatment. D. Induction of Saa1 and Saa2 gene expression in retinas of eyes with elevated intraocular pressure versus controls using semi-quantitative RT-PCR. Aliquots of RT-CR reactions were taken at 24, 29 and 34 cycles. Saa1 and Saa2 were increased in retinas of eyes exposed to IOP as compared with that of controls with the expression of Saa2 being the most prominent. Retinas were exposed to 3-weeks of elevated IOP during the 7-week post-injection treatment as those shown in Fig 1A. β -actin serves as the loading control.

Figure 4

Immunohistochemistry showing localization of proteins with IOP-induced altered gene expression in retinas of non-injected (A1–D1) and contralateral injected (A2–D2) eyes. Bcl2 expression level in the retina is decreased in the injected eyes (A2), whereas catalase (B2) and XIAP (C2) are increased. These changes in protein expression are localized to the ganglion cells of retinas of injected eyes. In particular, changes in expression of Bcl2, catalase and XIAP, are localized respectively, to the soma, dendritic processes (inner plexiform layer; IPL), and soma and dendritic processes, of ganglion neurons. In contrast, the serum amyloid A1/A2 (SAA1/SAA2) expression was present in scarce microglial cells spread throughout the retina (D2). These SAA1/SAA2-positive cells were not observed in retinas of non-injected eyes (D1). Retinas of injected eyes were exposed to 3-weeks of elevated IOP during the 7-week post-injection treatment as those shown in Fig 1A.

Figure 5

Detection of altered protein content in the vitreous of eyes with elevated IOP. A. Western blots of catalase (Cat) and XIAP protein levels in the vitreous of injected (Inj) and non-injected (Ctrl) contralateral eyes showed that these are increased only in eyes with elevated IOP. Hsc70 levels (lower panel) are comparable between the two groups of eyes and serves as a loading control. Injected eyes presented 3-weeks of elevated IOP during the 7-week post-injection treatment as those shown in Fig 1A. B. Densitometry analysis of changes the protein levels of catalase and XIAP in the vitreous of injected eye as compared with the control (shown in A). A 25-fold and a 15-fold average increase in the levels of catalase and XIAP were observed in the vitreous of injected eyes as compared with the controls (n=3). C. The vitreous presents no contamination with retina tissue. Western blot analysis of rhodopsin protein levels in the vitreous of injected and control contralateral eyes. The vitreous of injected (Inj) and non-injected (Ctrl) eyes present traces or no detectable rhodopsin, whereas this protein is highly abundant the retina. Note protein content of vitreous extracts loaded is 10-fold higher than the retina. D. Elevated IOP induces a significant decrease of the level of SAA in the serum. Data are presented as mean ± standard deviation (n=6).

Figure 6

The elevation of IOP is associated with a significant decrease in H₂O₂ in the retina. Elevated IOP induces a significant decrease of the level of H₂O₂ in the retina of injected eyes (Inj) compared to contralateral non-injected (Ctrl) eyes. Retinas of injected eyes were exposed to 3-weeks of elevated IOP during the 7-week post-injection treatment as those shown in Fig 1A. Data are presented as means ± standard deviation (n=3).