Deficits in mitochondrial TCA cycle and OXPHOS precede rod photoreceptor degeneration during chronic HIF activation.

BACKGROUND: Major retinal degenerative diseases, including age-related macular degeneration, diabetic retinopathy and retinal detachment, are associated with a local decrease in oxygen availability causing the formation of hypoxic areas affecting the photoreceptor (PR) cells. Here, we addressed the underlying pathological mechanisms of PR degeneration by focusing on energy metabolism during chronic activation of hypoxia-inducible factors (HIFs) in rod PR. METHODS: We used two-photon laser scanning microscopy (TPLSM) of genetically encoded biosensors delivered by adeno-associated viruses (AAV) to determine lactate and glucose dynamics in PR and inner retinal cells. Retinal layer-specific proteomics, in situ enzymatic assays and immunofluorescence studies were used to analyse mitochondrial metabolism in rod PRs during chronic HIF activation. RESULTS: PRs exhibited remarkably higher glycolytic flux through the hexokinases than neurons of the inner retina. Chronic HIF activation in rods did not cause overt change in glucose dynamics but an increase in lactate production nonetheless. Furthermore, dysregulation of the oxidative phosphorylation pathway (OXPHOS) and tricarboxylic acid (TCA) cycle in rods with an activated hypoxic response decelerated cellular anabolism causing shortening of rod photoreceptor outer segments (OS) before onset of cell degeneration. Interestingly, rods with deficient OXPHOS but an intact TCA cycle did not exhibit these early signs of anabolic dysregulation and showed a slower course of degeneration. CONCLUSION: Together, these data indicate an exceeding high glycolytic flux in rods and highlight the importance of mitochondrial metabolism and especially of the TCA cycle for PR survival in conditions of increased HIF activity.

Retinal regions shape human and murine Müller cell proteome profile and functionality.

The human macula is a highly specialized retinal region with pit-like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone- and rod-rich retinae from human and mice and identified different expression profiles of cone- and rod-associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell-derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region-specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo.

Retinal Layer Separation (ReLayS) method enables the molecular analysis of photoreceptor segments and cell bodies, as well as the inner retina.

Understanding the physiology of the retina, and especially of the highly polarized photoreceptors, is essential not only to broaden our knowledge of the processes required for normal vision, but also to develop effective therapies to prevent or slow retinal degenerative diseases. However, the molecular analysis of photoreceptors is a challenge due to the heterogeneity of the retinal tissue and the lack of easy and reliable methods for cell separation. Here we present the ReLayS method-a simple technique for the separation of photoreceptor segments (PS) containing both inner and outer segments, outer nuclear layer (ONL), and inner retina (InR) that contains the remaining retinal layers. The layer-specific material isolated from a mouse half-retina with the ReLayS method was sufficient for protein isolation and Western blotting or RNA isolation and real-time PCR studies. The separation of PS, ONL, and InR was successfully validated by Western blotting and real-time PCR using proteins and genes with known expression profiles within the retina. Furthermore, the separation of the PS from the ONL enabled the detection of light-driven translocation of transducin from the PS to the soma. ReLayS is a simple and useful method to address protein and possibly metabolites distribution in photoreceptor compartments in various situations including development, ageing, and degenerative diseases.

Stanniocalcin2, but Not Stanniocalcin1, Responds to Hypoxia in a HIF1-Dependent Manner in the Retina.

The quest for neuroprotective factors that can prevent or slow down the progression of retinal degeneration is still ongoing. Acute hypoxic stress has been shown to provide transient protection against subsequent damage in the retina. Stanniocalcins - STC1 and STC2 - are secreted glycoproteins that are hypoxia-regulated and were shown to be cytoprotective in various in vitro studies. Hence, we investigated the expression of stanniocalcins in the normal, degenerating and hypoxic retina. We show that the expression of Stc1 and Stc2 in the retina was detectable as early as postnatal day 10 and persisted during aging. Retinal expression of Stc2, but not Stc1, was induced in mice in an in vivo model of acute hypoxia and a genetic model of chronic hypoxia. Furthermore, we show that HIF1, not HIF2, is responsible for regulating Stc2 in cells with the molecular response to hypoxia activated due to the absence of von Hippel Lindau protein. Surprisingly, Stc2 was not normally expressed in photoreceptors but in the inner retina, as shown by laser capture microdissection and immunofluorescence data. The expression of both Stc1 and Stc2 remained unchanged in the degenerative retina with an almost complete loss of photoreceptors, confirming their expression in the inner retina. However, the absence of either Stc1 or Stc2 had no effect on retinal architecture, as was evident from retinal morphology of the respective knockout mice. Taken together our data provides evidence for the differential regulation of STC1 and STC2 in the retina and the prospect of investigating STC2 as a retinal neuroprotective factor.

Increased H3K27 trimethylation contributes to cone survival in a mouse model of cone dystrophy.

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite individual IRDs (Inherited retinal disease) being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6ccpfl1 mouse model of achromatopsia, a cone-specific vision loss IRD (Inherited retinal disease), to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD (Inherited retinal disease)-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in developing a broad class of novel therapies to slow cone degeneration.

HIF1 and DROSHA are involved in MMACHC repression in hypoxia.

The MMACHC gene encodes for an enzyme involved in intracellular vitamin B12 metabolism, and autosomal recessive defects in MMACHC represent the most common disorder of intracellular vitamin B12 metabolism. Recent studies have identified increased levels of reactive oxygen species in cells and tissues with MMACHC dysfunction, suggesting a role for oxidative stress in disease. To investigate the link between oxidative stress and MMACHC, we exposed mice as well as human and mouse cells to hypoxia, and found significant repression of MMACHC in all investigated tissues (retina, eyecup, liver, kidney) and cell lines (HeLa, ARPE-19, human and mouse fibroblasts, 661W). Furthermore, in HeLa cells, we found transcriptional repression already at 5% oxygen, which was stable during prolonged hypoxia up to 5 days, and a return of MMACHC transcripts to normal levels only 24 h after reoxygenation. This hypoxia-induced downregulation of MMACHC was not due to altered function of the known MMACHC controlling transcription factor complex HCFC1/THAP11/ZNF143. Using in vitro RNA interference against hypoxia-induced transcription factors (HIF1A, HIF2A and REST) as well as the microRNA transcription machinery (DROSHA), we observed release of hypoxia-dependent downregulation of MMACHC expression by HIF1A and DROSHA knockdowns, whose combined effect was additive. Together, these results strongly indicate that MMACHC is a hypoxia-regulated gene whose downregulation appears to be partially mediated through both hypoxia-induced transcription factor and microRNA machinery. These findings suggest that oxidative stress could impair vitamin B12 metabolism by repression of MMACHC in healthy as well as in diseased individuals.

Single-cell RNA sequencing of the retina in a model of retinitis pigmentosa reveals early responses to degeneration in rods and cones.

BACKGROUND: In inherited retinal disorders such as retinitis pigmentosa (RP), rod photoreceptor-specific mutations cause primary rod degeneration that is followed by secondary cone death and loss of high-acuity vision. Mechanistic studies of retinal degeneration are challenging because of retinal heterogeneity. Moreover, the detection of early cone responses to rod death is especially difficult due to the paucity of cones in the retina. To resolve heterogeneity in the degenerating retina and investigate events in both types of photoreceptors during primary rod degeneration, we utilized droplet-based single-cell RNA sequencing in an RP mouse model, rd10. RESULTS: Using trajectory analysis, we defined two consecutive phases of rod degeneration at P21, characterized by the early transient upregulation of Egr1 and the later induction of Cebpd. EGR1 was the transcription factor most significantly associated with the promoters of differentially regulated genes in Egr1-positive rods in silico. Silencing Egr1 affected the expression levels of two of these genes in vitro. Degenerating rods exhibited changes associated with metabolism, neuroprotection, and modifications to synapses and microtubules. Egr1 was also the most strongly upregulated transcript in cones. Its upregulation in cones accompanied potential early respiratory dysfunction and changes in signaling pathways. The expression pattern of EGR1 in the retina was dynamic during degeneration, with a transient increase of EGR1 immunoreactivity in both rods and cones during the early stages of their degenerative processes. CONCLUSION: Our results identify early and late changes in degenerating rd10 rod photoreceptors and reveal early responses to rod degeneration in cones not expressing the disease-causing mutation, pointing to mechanisms relevant for secondary cone degeneration. In addition, our data implicate EGR1 as a potential key regulator of early degenerative events in rods and cones, providing a potential broad target for modulating photoreceptor degeneration.

Circadian regulation of vertebrate cone photoreceptor function.

Eukaryotes generally display a circadian rhythm as an adaption to the reoccurring day/night cycle. This is particularly true for visual physiology that is directly affected by changing light conditions. Here we investigate the influence of the circadian rhythm on the expression and function of visual transduction cascade regulators in diurnal zebrafish and nocturnal mice. We focused on regulators of shut-off kinetics such as Recoverins, Arrestins, Opsin kinases, and Regulator of G-protein signaling that have direct effects on temporal vision. Transcript as well as protein levels of most analyzed genes show a robust circadian rhythm-dependent regulation, which correlates with changes in photoresponse kinetics. Electroretinography demonstrates that photoresponse recovery in zebrafish is delayed in the evening and accelerated in the morning. Functional rhythmicity persists in continuous darkness, and it is reversed by an inverted light cycle and disrupted by constant light. This is in line with our finding that orthologous gene transcripts from diurnal zebrafish and nocturnal mice are often expressed in an anti-phasic daily rhythm.

Absence of MMACHC in peripheral retinal cells does not lead to an ocular phenotype in mice.

Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B12 metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.

HDAC inhibition ameliorates cone survival in retinitis pigmentosa mice.

Cone photoreceptor cell death in inherited retinal diseases, such as Retinitis Pigmentosa (RP), leads to the loss of high acuity and color vision and, ultimately to blindness. In RP, a vast number of mutations perturb the structure and function of rod photoreceptors, while cones remain initially unaffected. Extensive rod loss in advanced stages of the disease triggers cone death by a mechanism that is still largely unknown. Here, we show that secondary cone cell death in animal models for RP is associated with increased activity of histone deacetylates (HDACs). A single intravitreal injection of an HDAC inhibitor at late stages of the disease, when the majority of rods have already degenerated, was sufficient to delay cone death and support long-term cone survival in two mouse models for RP, affected by mutations in the phosphodiesterase 6b gene. Moreover, the surviving cones remained light-sensitive, leading to an improvement in visual function. RNA-seq analysis of protected cones demonstrated that HDAC inhibition initiated multi-level protection via regulation of different pro-survival pathways, including MAPK, PI3K-Akt, and autophagy. This study suggests a unique opportunity for targeted pharmacological protection of secondary dying cones by HDAC inhibition and creates hope to maintain vision in RP patients even in advanced disease stages.

PEDF deficiency increases the susceptibility of rd10 mice to retinal degeneration.

The SERPINF1 gene encodes pigment epithelium-derived factor (PEDF), a member of the serpin superfamily with neurotrophic and antiangiogenic properties in the retina. We hypothesized that absence of PEDF would lead to increased stress-associated retinal degeneration in Serpinf1 null mice. Accordingly, using a Serpinf1 null mouse model, we investigated the impact of PEDF absence on retinal morphology, and susceptibility to induced and inherited retinal degeneration. We studied the pattern of Serpinf1 expression in the mouse retina layers. PEDF protein was detected by western blotting. Transmission electron microscopy was performed on mouse retina. Serpinf1 null mice and wild type littermates were injected with NaIO3 (30 mg/kg body weight) intraperitonially. At post-injection day 1, 3, 4, 6 and 8 mice were euthanized, and eyes were enucleated. Serpinf1 null and rd10 double mutant mice were generated and their eyes enucleated at different time points from post-natal day 15 to post-natal day 28. Enucleated eyes were processed for hematoxylin and eosin staining and histopathological evaluations. We found that Serpinf1 was expressed in the retinal pigment epithelium, in the inner nuclear layer and in the ganglion cell layer, but undetectable in the outer nuclear layer of wild type mice. Plasma PEDF protein levels were undetectable in Serpinf1 null animals. RPE atrophy and retinal thinning were observed in NaIO3-treated wild type mice that progressed with time post-injection. NaIO3-treated Serpinf1 null mice showed comparatively better retinal morphology than wild type mice at day 4 post-injection. However, the absence of PEDF in Serpinf1 null x rd10 mice increased the susceptibility to retinal degeneration relative to that of rd10 mice. We concluded that histopathological evaluation of retinas lacking PEDF showed that removal of the Serpinf1 gene may activate PEDF-independent compensatory mechanisms to protect the retina against oxidative stress, while it increases the susceptibility to degenerate the retina in inherited retinal degeneration models.

The Expression of Decidual Protein Induced by Progesterone (DEPP) is Controlled by Three Distal Consensus Hypoxia Responsive Element (HRE) in Hypoxic Retinal Epithelial Cells.

Hypoxia affects the development and/or progression of several retinopathies. Decidual protein induced by progesterone (DEPP) has been identified as a hypoxia-responsive gene that may be part of cellular pathways such as autophagy and connected to retinal diseases. To increase our understanding of DEPP regulation in the eye, we defined its expression pattern in mouse and human retina and retinal pigment epithelium (RPE). Interestingly, DEPP expression was increased in an age-dependent way in the central human RPE. We showed that DEPP was regulated by hypoxia in the mouse retina and eyecup and that this regulation was controlled by hypoxia-inducible transcription factors 1 and 2 (HIF1 and HIF2). Furthermore, we identified three hypoxia response elements (HREs) about 3.5 kb proximal to the transcriptional start site that were responsible for hypoxic induction of DEPP in a human RPE cell line. Comparative genomics analysis suggested that one of the three HREs resides in a highly conserved genomic region. Collectively, we defined the molecular elements controlling hypoxic induction of DEPP in an RPE cell line, and provided evidence for an enrichment of DEPP in the aged RPE of human donors. This makes DEPP an interesting gene to study with respect to aging and age-related retinal pathologies.

HDAC Inhibition Prevents Primary Cone Degeneration Even After the Onset of Degeneration.

Cone photoreceptor loss is the main cause of color blindness and loss of visual acuity in patients suffering from inherited cone dystrophies. Despite the crucial role of cones in everyday life, knowledge on mechanisms of cone cell death and the identification of potential targets for the preservation or delay of cone loss are scarce. Recent findings have shown that excessive histone deacetylase (HDAC) activity is associated with both primary rod and primary cone degeneration. Importantly, pharmacological inhibition of HDAC activity in vivo at the onset of cone degeneration offers a prolonged protection of cones in a mouse model of inherited cone degeneration (cpfl1). In this study, we evaluated the potential of trichostatin A (TSA), a pan-HDAC inhibitor, to prevent cone cell death at a later stage of degeneration in the cpfl1 model. We demonstrate that a single intravitreal TSA injection protected the cpfl1 cones even when administered after the onset of degeneration. In addition, the TSA treatment significantly improved aberrant cone nucleokinesis present in the cpfl1 retina. These results highlight the feasibility of targeted cone neuroprotection in vivo even at later disease stages of inherited cone dystrophies.

Hypoxia-Regulated MicroRNAs in the Retina.

The retina is one of the tissues with the highest metabolic activity in the body, and the energy-demanding photoreceptors require appropriate oxygen levels for photo- and neurotransduction. Accumulating evidence suggests that age-related changes in the retina may reduce oxygen supply to the photoreceptors and trigger a chronic hypoxic response. A detailed understanding of the molecular response to hypoxia is crucial, as hindered oxygen delivery may contribute to the development and progression of retinal pathologies such as age-related macular degeneration (AMD). Important factors in the cellular response to hypoxia are microRNAs (miRNAs), which are small, noncoding RNAs that posttranscriptionally regulate gene expression by binding to mRNA transcripts. Here, we discuss the potential role of hypoxia-regulated miRNAs in connection to retinal pathologies.

Hif1a and Hif2a can be safely inactivated in cone photoreceptors.

Impaired tissue oxygenation results in hypoxia and leads to the activation of hypoxia-inducible transcription factors (HIF). A chronic, HIF-triggered molecular response to hypoxia may be an important factor in the etiology of age-related macular degeneration (AMD) and is likely activated before any clinical manifestation of the disease. Thus, HIF1 and HIF2 recently emerged as potential therapeutic targets for AMD. To address and evaluate potential consequences of anti-HIF therapies for retinal physiology and function, we generated mouse lines that have Hif1a, or both Hif1a and Hif2a ablated specifically in cone photoreceptors. The knockdown of Hifs in cones did not cause detectable pathological alterations such as loss of cone photoreceptors, retinal degeneration or abnormalities of the retinal vasculature, had no impact on retinal function and resulted in a similar tolerance to hypoxic exposure. Our data indicate that HIF transcription factors are dispensable for maintaining normal cone function and survival in retinas of adult mice. This study provides the groundwork necessary to establish safety profiles for strategies aiming at antagonizing HIF1A and HIF2A function in cone photoreceptors for the treatment of retinal degenerative diseases that involve a hypoxic component such as AMD.

Correction to: Retinal pathology in experimental optic neuritis is characterized by retrograde degeneration and gliosis.

In the original publication of this article [1], Fig. 10 contained two panels "C" as panel "F" was accidentally omitted. The incorrect (Fig. 1) and correct (Fig. 2) versions are published in this correction article.

Systemic knockout of Tspo in mice does not affect retinal morphology, function and susceptibility to degeneration.

Translocator protein (18 kDa) (TSPO) is a mitochondrial protein expressed by reactive microglia and astrocytes at the site of neuronal injury. Although TSPO function has not been fully determined, synthetic TSPO ligands have beneficial effects on different pathologies of the central nervous system, including the retina. Here, we studied the pattern of Tspo expression in the aging human retina and in two mouse models of retinal degeneration. Using a newly generated Tspo-KO mouse, we investigated the impact of the lack of TSPO on retinal morphology, function and susceptibility to degeneration. We show that TSPO was expressed in both human and mouse retina and retinal pigment epithelium (RPE). Tspo was induced in the mouse retina upon degeneration, but constitutively expressed in the RPE. Similarly, TSPO expression levels in healthy human retina and RPE were not differentially regulated during aging. Tspo-KO mice had normal retinal morphology and function up to 48 weeks of age. Photoreceptor loss caused either by exposure to excessive light levels or by a mutation in the phosphodiesterase 6b gene was not affected by the absence of Tspo. The reactivity states of retinal mononuclear phagocytes following light-damage were comparable in Tspo-KO and control mice. Our data suggest that lack of endogenous TSPO does not directly influence the magnitude of photoreceptor degeneration or microglia activation in these two models of retinal degeneration. We therefore hypothesize that the interaction of TSPO with its ligands may be required to modulate disease progression.

Retinal pathology in experimental optic neuritis is characterized by retrograde degeneration and gliosis.

The exact mechanisms and temporal sequence of neurodegeneration in multiple sclerosis are still unresolved. The visual pathway including its unmyelinated retinal axons, can serve as a prototypic model of neurodegeneration in experimental optic neuritis. We conducted a longitudinal study combining retinal imaging through optical coherence tomography (OCT) with immunohistochemical analyses of retinal and optic nerve tissue at various time points in experimental autoimmune encephalomyelitis (EAE).Inner retinal layer (IRL) thickness was measured in 30 EAE and 14 healthy control C57BL/6 J mice using OCT. Distribution of marker proteins was assessed by immunofluorescence staining and retinal mRNA levels were assayed using real-time PCR. Histological morphology was evaluated on light and electron microscopy images.Signs of inflammatory edema 11 days post immunisation coincided with IRL thickening, while neuro-axonal degeneration throughout the disease course contributed to IRL thinning observed after 20 days post immunisation. Retinal pathology, including axonal transport impairment, was observed early, prior to cellular infiltration (i.e. T-cells) in the optic nerve 11 days post immunisation. Yet, the effects of early retinal damage on OCT-derived readouts were outweighed by the initial inflammatory edema. Early microglial activation and astrocytosis was detected in the retina prior to retinal ganglion cell loss and persisted until 33 days post immunisation. Müller cell reactivity (i.e. aquaporin-4 and glutamine synthetase decrease) presented after 11 days post immunisation in the IRL. Severe neuro-axonal degeneration was observed in the optic nerve and retina until 33 days post immunisation.Initial signs of retinal pathology subsequent to early glial activity, suggests a need for prophylactic treatment of optic neuritis. Following early inflammation, Müller cells possibly respond to retinal pathology with compensatory mechanisms. Although the majority of the IRL damage observed is likely due to retrograde degeneration following optic neuritis, initial pathology, possibly due to gliosis, may contribute further to IRL thinning. These results add morphological substrate to our OCT findings. The extent and rapid onset of axonal and neuronal damage in this model appears relevant for testing interventions scaled to human optic neuritis.

Light stress affects cones and horizontal cells via rhodopsin-mediated mechanisms.

Retinal degenerations are a major cause of blindness in human patients. The identification of endogenous mechanisms involved in neurodegeneration or neuroprotection helps to understand the response of the retina to stress and provides essential information not only for basic retinal physiology but also for defining molecular targets for neuroprotective strategies. Here we used excessive light exposure as a model system to study mechanisms of photoreceptor degeneration in mice. Using one wild type and four genetically modified mouse strains, we demonstrate that light exposure resulted not only in the degeneration of rods but also in an early but transient repression of several cone-specific genes, in a reversible hyperreflectivity of the outer retina including the outer plexiform layer, and in the loss of horizontal cells. The effects on cones, horizontal cells and the inner retina depended on light absorption by rhodopsin and, at least partially, on leukemia inhibitory factor. This demonstrates the existence of intercellular communication routes that transduce rod stress to other cells, likely to provide support for photoreceptors and increase cell survival in the injured retina.

A chronic hypoxic response in photoreceptors alters the vitreous proteome in mice.

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 (rodΔVhl) or cones (all-coneΔVhl). 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 rodΔVhl and in all-coneΔVhl 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 rodΔVhl and all-coneΔVhl 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.