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Reduced oxygenation of the outer retina in the aging eye may activate a chronic hypoxic response in RPE and photoreceptor cells and is considered as a risk factor for the development of age-related macular degeneration (AMD). In mice, a chronically active hypoxic response in the retinal pigment epithelium (RPE) or photoreceptors leads to age-dependent retinal degeneration. To identify proteins that may serve as accessible markers for a chronic hypoxic insult to photoreceptors, we used proteomics to determine the protein composition of the vitreous humor in genetically engineered mice that lack the von Hippel-Lindau tumor suppressor (Vhl) specifically in rods (rod ΔVhl ) or cones (all-cone ΔVhl ). Absence of VHL leads to constitutively active hypoxia-inducible transcription factors (HIFs) and thus to a molecular response to hypoxia even in normal room air. To discriminate between the consequences of a local response in photoreceptors and systemic hypoxic effects, we also evaluated the vitreous proteome of wild type mice after exposure to acute hypoxia. 1'043 of the identified proteins were common to all three hypoxia models. 257, 258 and 356 proteins were significantly regulated after systemic hypoxia, in rod ΔVhl and in all-cone ΔVhl mice, respectively, at least at one of the analyzed time points. Only few of the regulated proteins were shared by the models indicating that the vitreous proteome is differentially affected by systemic hypoxia and the rod or cone-specific hypoxic response. Similarly, the distinct protein compositions in the individual genetic models at early and late time points suggest regulated, cell-specific and time-dependent processes. Among the proteins commonly regulated in the genetic models, guanylate binding protein 2 (GBP2) showed elevated levels in the vitreous that were accompanied by increased mRNA expression in the retina of both rod ΔVhl and all-cone ΔVhl mice. We hypothesize that some of the differentially regulated proteins at early time points may potentially be used as markers for the detection of a chronic hypoxic response of photoreceptors., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: In contrast to neovascular AMD (nAMD), no treatment option exists for dry AMD. Hence, the identification of specific biomarkers is required to facilitate diagnosis and therapy of dry AMD., Methods: The proteome of 34 vitreous humor samples (dry AMD: n = 6; nAMD: n = 10; proliferative diabetic retinopathy [PDR]: n = 9; epiretinal membrane [ERM]: n = 9) was analyzed by liquid chromatography coupled mass spectrometry. Then, label-free relative quantification of dry AMD, nAMD, and PDR relative to ERM, which was defined as the reference group, was performed. Application of a bioinformatics pipeline further analyzed the vitreous proteome by cluster and gene set enrichment analysis. A selection of differentially regulated proteins was validated by ELISA., Results: A total of 677 proteins were identified in the vitreous of the four patient groups and quantified relatively to ERM. Different clusters of regulated proteins for each patient group were identified and showed characteristic enrichment of specific pathways including "oxidative stress" for dry AMD, "focal adhesion" for nAMD, and "complement and coagulation cascade" for PDR patients. We identified cholinesterase (CHLE) to be specifically upregulated in dry AMD and ribonuclease (pancreatic; RNAS1) together with serine carboxypeptidase (probable; CPVL) to be upregulated in both forms of AMD., Conclusions: The described pathways specific for the different patient groups and the identification of characteristic differentially regulated proteins provide a first step toward the definition of biomarkers for dry AMD. The presented data will facilitate the investigation of mechanistic connections of proteins to the respective disease.

Mutations in the elongation of very long chain fatty acid 4 (ELOVL4) gene cause Stargardt macular dystrophy 3 (STGD3), a rare, juvenile-onset, autosomal dominant form of macular degeneration. Although several mouse models have already been generated to investigate the link between the three identified disease-causing mutations in the ELOVL4 gene, none of these models recapitulates the early-onset cone photoreceptor cell death observed in the macula of STGD3 patients. To address this specifically, we investigated the effect of mutant ELOVL4 in a mouse model with an all-cone retina. Hence, we bred mice carrying the heterozygously mutated Elovl4 gene on the R91W;Nrl-/- all-cone background and analyzed the retinal lipid composition, morphology, and function over the course of 1 year. We observed a reduction of total phosphatidylcholine-containing very long chain-polyunsaturated fatty acids (PC-VLC-PUFAs) by 39% in the R91W;Nrl-/-;Elovl4 mice already at 6 weeks of age with a pronounced decline of the longest forms of PC-VLC-PUFAs. Total levels of shorter-chain fatty acids (< C26) remained unaffected. However, this reduction in PC-VLC-PUFA content in the all-cone retina had no impact on morphology or function and did not accelerate retinal degeneration in the R91W;Nrl-/-;Elovl4 mice. Taken together, mutations in the ELOVL4 gene lead to cone degeneration in humans, whereas mouse models expressing the mutant Elovl4 show predominant rod degeneration. The lack of a phenotype in the all-cone retina expressing the mutant form of the protein supports the view that aberrant function of ELOVL4 is especially detrimental for rods in mice and suggests a more subtle role of VLC-PUFAs for cone maintenance and survival.