Retinal proteomics of experimental glaucoma model reveal intraocular pressure‐induced mediators of neurodegenerative changes

Abstract

Current evidence suggests that exposure to chronically induced intraocular pressure (IOP) leads to neurodegenerative changes in the inner retina. This study aimed to determine retinal proteomic alterations in a rat model of glaucoma and compared findings with human retinal proteomics changes in glaucoma reported previously. We developed an experimental glaucoma rat model by subjecting the rats to increased IOP (9.3 ± 0.1 vs 20.8 ± 1.6 mm Hg) by weekly microbead injections into the eye (8 weeks). The retinal tissues were harvested from control and glaucomatous eyes and protein expression changes analysed using a multiplexed quantitative proteomics approach (TMT-MS3). Immunofluorescence was performed for selected protein markers for data validation. Our study identified 4304 proteins in the rat retinas. Out of these, 139 proteins were downregulated (≤0.83) while the expression of 109 proteins was upregulated (≥1.2-fold change) under glaucoma conditions (P ≤ .05). Computational analysis revealed reduced expression of proteins associated with glutathione metabolism, mitochondrial dysfunction/oxidative phosphorylation, cytoskeleton, and actin filament organisation, along with increased expression of proteins in coagulation cascade, apoptosis, oxidative stress, and RNA processing. Further functional network analysis highlighted the differential modulation of nuclear receptor signalling, cellular survival, protein synthesis, transport, and cellular assembly pathways. Alterations in crystallin family, glutathione metabolism, and mitochondrial dysfunction associated proteins shared similarities between the animal model of glaucoma and the human disease condition. In contrast, the activation of the classical complement pathway and upregulation of cholesterol transport proteins were exclusive to human glaucoma. These findings provide insights into the Mehdi Mirzaei, Vivek K. Gupta, and Nitin Chitranshi contributed equally to this study. neurodegenerative mechanisms that are specifically affected in the retina in response to chronically elevated IOP.

Proteomic Comparison of Aged and OPTN E50K Retina in the Development of Normal Tension Glaucoma

“…As for crystallin proteins, involved in eye development and protein folding, their levels were decreased in both NTG and AD mice [26,27,[60][61][62][63]. Indeed, a reduction in abundance was observed in previous studies for 7 such proteins in association with high IOP [26,64], as well as here for young OPTN E50K mice. In particular, 2 crystallin proteins within those 7, Crybb3 and Cryab, were also found to have signi cantly decreased in AD mice retina [27], further supporting ndings highlighting biomolecular similarities between glaucoma and AD.…”

Differential Retinal Protein Expression in Primary and Secondary Retinal Ganglion Cell Degeneration Identified by Integrated SWATH and Target-Based Proteomics

“…In addition to significant changes in filament organization, our study also demonstrated that alterations in glutathione metabolism and mitochondrial dysfunction-associated proteins, activation of the coagulation cascade, and upregulation of cholesterol transport proteins exhibited similarities with the rat model of glaucoma analyzed using a multiplexed quantitative proteomics approach [33]. Numerous regulated proteins shared by the two rat models of pONT and IOP elevation via microbead injection include AHNAK, APOE, FGB, KNG1, NDUFA2, GSTM5, and members of S100, crystallin, CPLX, and ANXA families, providing evidence that the mechanisms underlying the progressive RGC degeneration after pONT injury are relevant to glaucoma.…”

Antibody and Protein Profiles in Glaucoma: Screening of Biomarkers and Identification of Signaling Pathways

“…Second, the retina of the donors was separated from other eye tissues after death, thus contamination, especially with lens tissue, cannot be excluded. In two different experimental rat glaucoma models (both in Spraque Dawley rats) with chronic elevated IOP, once by microbead injection (MB) and measurement of protein changes after eight weeks [ 36 ] and the other through episcleral vein occlusion (EVO) and measurement after five weeks. A down-regulation of many crystallins of all subfamilies could also be detected here in retinal tissue [ 37 ].…”

“…Additionally, other proteins involved in cytoskeleton organization and protein folding are found to be differentially expressed in the glaucomatous retina. Three types of tropomyosins (TPM1, TPM3, and TPM4), the RAS GTPase-activating-like protein IQGAP2 (IQGAP2), and the neuroblast differentiation-associated protein AHNAK (AHNAK) were found to be down-regulated in human and rat retina [ 36 ]. Contrary, IQGAP2 protein level was found to be highly up-regulated in EVO-induced rat models [ 37 ].…”