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.

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.

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.

OCRL deficiency impairs endolysosomal function in a humanized mouse model for Lowe syndrome and Dent disease.

Mutations in OCRL encoding the inositol polyphosphate 5-phosphatase OCRL (Lowe oculocerebrorenal syndrome protein) disrupt phosphoinositide homeostasis along the endolysosomal pathway causing dysfunction of the cells lining the kidney proximal tubule (PT). The dysfunction can be isolated (Dent disease 2) or associated with congenital cataracts, central hypotonia and intellectual disability (Lowe syndrome). The mechanistic understanding of Dent disease 2/Lowe syndrome remains scarce due to limitations of animal models of OCRL deficiency. Here, we investigate the role of OCRL in Dent disease 2/Lowe syndrome by using OcrlY/- mice, where the lethal deletion of the paralogue Inpp5b was rescued by human INPP5B insertion, and primary culture of proximal tubule cells (mPTCs) derived from OcrlY/- kidneys. The OcrlY/- mice show muscular defects with dysfunctional locomotricity and present massive urinary losses of low-molecular-weight proteins and albumin, caused by selective impairment of receptor-mediated endocytosis in PT cells. The latter was due to accumulation of phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 in endolysosomes, driving local hyper-polymerization of F-actin and impairing trafficking of the endocytic LRP2 receptor, as evidenced in OcrlY/- mPTCs. The OCRL deficiency was also associated with a disruption of the lysosomal dynamic and proteolytic activity. Partial convergence of disease-pathways and renal phenotypes observed in OcrlY/- and Clcn5Y/- mice suggest shared mechanisms in Dent diseases 1 and 2. These studies substantiate the first mouse model of Lowe syndrome and give insights into the role of OCRL in cellular trafficking of multiligand receptors. These insights open new avenues for therapeutic interventions in Lowe syndrome and Dent disease.

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.

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.

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.

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.

Exploring experimental autoimmune optic neuritis using multimodal imaging.

BACKGROUND: Neuro-axonal injury is a key contributor to non-reversible long-term disability in multiple sclerosis (MS). However, the underlying mechanisms are not yet fully understood. Visual impairment is common among MS patients, in which episodes of optic neuritis (ON) are often followed by structural retinal damage and sustained functional impairment. Alterations in the optic nerve and retina have also been described in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. Thus, investigating structural anterior visual pathway damage may constitute a unique model for assessing mechanisms and temporal sequence of neurodegeneration in MS. We used a multimodal imaging approach utilizing optical coherence tomography (OCT) and diffusion tensor imaging (DTI) to explore the mechanisms and temporal dynamics of visual pathway damage in the animal model of MS. METHODS: 7 EAE-MOG35-55 and 5 healthy female C57BL/6J mice were used in this study. Ganglion cell complex (GCC) thickness was derived from an OCT volume scan centred over the optic nerve head, while the structure of the optic nerve and tracts was assessed from DTI and co-registered T2-weighted sequences performed on a 7T MRI scanner. Data was acquired at baseline, disease onset, peak of disease and recovery. Linear mixed effect models were used to account for intra-subject, inter-eye dependencies, group and time point. Correlation analyses assessed the relationship between GCC thickness and DTI parameters. Immunofluorescence staining of retina and optic nerve sections was used to assess distribution of marker proteins for microglia and neurodegeneration (nerve filaments). RESULTS: In EAE mice, a significant increase in GCC thickness was observed at disease onset (p < 0.001) followed by a decrease at recovery (p < 0.001) compared to controls. The EAE group had significant GCC thinning at recovery compared to all other time points (p < 0.001 for each). Signal increase on T2-weighted images around the optic nerves indicative of inflammation was seen in most of the EAE mice but in none of the controls. A significant decrease in axial diffusivity (AD) and increase in radial diffusivity (RD) values in EAE optic nerves (AD: p = 0.02, RD: p = 0.01) and tract (AD: p = 0.02, RD: p = 0.006) was observed compared to controls. GCC at recovery was positively correlated with AD (optic nerve: rho = 0.74, p = 0.04, optic tract: rho = 0.74, p = 0.04) and negatively correlated with RD (optic nerve: rho = -0.80, p = 0.02, optic tract: rho = -0.75, p = 0.04). Immunofluorescence analysis indicated the presence of activated microglia in the retina and optic nerves in addition to astrocytosis and axonal degeneration in the optic nerve of EAE mice. CONCLUSION: OCT detected GCC changes in EAE may resemble what is observed in MS-related acute ON: an initial phase of swelling (indicative of inflammatory edema) followed by a decrease in thickness over time (representative of neuro-axonal degeneration). In line with OCT findings, DTI of the visual pathway identifies EAE induced pathology (decreased AD, and increased RD). Immunofluorescence analysis provides support for inflammatory pathology and axonal degeneration. OCT together with DTI can detect retinal and optic nerve damage and elucidate to the temporal sequence of neurodegeneration in this rodent model of MS in vivo.

Cone Genesis Tracing by the Chrnb4-EGFP Mouse Line: Evidences of Cellular Material Fusion after Cone Precursor Transplantation.

The cone function is essential to mediate high visual acuity, color vision, and daylight vision. Inherited cone dystrophies and age-related macular degeneration affect a substantial percentage of the world population. To identify and isolate the most competent cells for transplantation and integration into the retina, cone tracing during development would be an important added value. To that aim, the Chrnb4-EGFP mouse line was characterized throughout retinogenesis. It revealed a sub-population of early retinal progenitors expressing the reporter gene that is progressively restricted to mature cones during retina development. The presence of the native CHRNB4 protein was confirmed in EGFP-positive cells, and it presents a similar pattern in the human retina. Sub-retinal transplantations of distinct subpopulations of Chrnb4-EGFP-expressing cells revealed the embryonic day 15.5 high-EGFP population the most efficient cells to interact with host retinas to provoke the appearance of EGFP-positive cones in the photoreceptor layer. Importantly, transplantations into the DsRed retinas revealed material exchanges between donor and host retinas, as >80% of transplanted EGFP-positive cones also were DsRed positive. Whether this cell material fusion is of significant therapeutic advantage requires further thorough investigations. The Chrnb4-EGFP mouse line definitely opens new research perspectives in cone genesis and retina repair.

The Role of Hypoxia, Hypoxia-Inducible Factor (HIF), and VEGF in Retinal Angiomatous Proliferation.

In industrialized countries, age-related macular degeneration (AMD) is the leading cause of blindness in elderly people. Hallmarks of the non-neovascular (dry) form of AMD are the formation of drusen and geographic atrophy, whereas the exudative (wet) form of the disease is characterized by invading blood vessels. In retinal angiomatous proliferation (RAP), a special form of wet AMD, intraretinal vessels grow from the deep plexus into the subretinal space. Little is known about the mechanisms leading to intraretinal neovascularization, but age-related changes such as reduction of choroidal blood flow, accumulation of drusen, and thickening of the Bruch's membrane may lead to reduced oxygen availability in photoreceptors. Such a chronic hypoxic situation may induce several cellular response pathways including the stabilization of hypoxia-inducible factors (HIFs) and the production of angiogenic factors, such as vascular endothelial growth factor (VEGF). Here, we discuss the potential contribution of hypoxia and HIFs in RAP disease pathology and in some mouse models for subretinal neovascularization.

Pharmacological Amelioration of Cone Survival and Vision in a Mouse Model for Leber Congenital Amaurosis.

UNLABELLED: RPE65, an abundant membrane-associate protein in the retinal pigment epithelium (RPE), is a key retinoid isomerase of the visual cycle necessary for generating 11-cis-retinal that functions not only as a molecular switch for activating cone and rod visual pigments in response to light stimulation, but also as a chaperone for normal trafficking of cone opsins to the outer segments. Many mutations in RPE65 are associated with Leber congenital amaurosis (LCA). A R91W substitution, the most frequent LCA-associated mutation, results in a severe decrease in protein level and enzymatic activity of RPE65, causing cone opsin mislocalization and early cone degeneration in the mutation knock-in mouse model of LCA. Here we show that R91W RPE65 undergoes ubiquitination-dependent proteasomal degradation in the knock-in mouse RPE due to misfolding. The 26S proteasome non-ATPase regulatory subunit 13 mediated degradation specifically of misfolded R91W RPE65. The mutation disrupted membrane-association and colocalization of RPE65 with lecithin:retinol acyltransferase (LRAT) that provides the hydrophobic substrate for RPE65. Systemic administration of sodium 4-phenylbutyrate (PBA), a chemical chaperone, increased protein stability, enzymatic activity, membrane-association, and colocalization of R91W RPE65 with LRAT. This rescue effect increased synthesis of 11-cis-retinal and 9-cis-retinal, a functional iso-chromophore of the visual pigments, led to alleviation of S-opsin mislocalization and cone degeneration in the knock-in mice. Importantly, PBA-treatment also improved cone-mediated vision in the mutant mice. These results indicate that PBA, a U.S. Food and Drug Administration-approved safe oral medication, may provide a noninvasive therapeutic intervention that delays daylight vision loss in patients with RPE65 mutations. SIGNIFICANCE STATEMENT: LCA is a severe early onset retinal dystrophy. Recent clinical trials of gene therapy have implicated the need of an alternative or combination therapy to improve cone survival and function in patients with LCA caused by RPE65 mutations. Using a mouse model carrying the most frequent LCA-associated mutation (R91W), we found that the mutant RPE65 underwent ubiquitination-dependent proteasomal degradation due to misfolding. Treatment of the mice with a chemical chaperone partially corrected stability, enzymatic activity, and subcellular localization of R91W RPE65, which was also accompanied by improvement of cone survival and vision. These findings identify an in vivo molecular pathogenic mechanism for R91W mutation and provide a feasible pharmacological approach that can delay vision loss in patients with RPE65 mutations.

The Consequences of Hypomorphic RPE65 for Rod and Cone Photoreceptors.

RPE65 is essential for both rod- and cone-mediated vision. So far, more than 120 disease-associated mutations have been identified in the human RPE65 gene. Differential clinical manifestations suggested that some patients suffer from null mutations while others retain residual RPE65 activity and some useful vision. To understand the mechanism of retinal degeneration or dysfunction caused by such hypomorphic RPE65 alleles, we generated an Rpe65 (R91W) knock-in mouse (R91W) that expresses a mutant RPE65 protein with reduced function. Data obtained suggested that the R91W mouse is highly suitable to study the impact of RPE65 insufficiency on rod pathophysiology. To study the impact on cones, we combined the R91W with the Nrl (-/-) mouse that develops an all-cone retina. Here we summarize the consequences of hypomorphic RPE65 function (reduced 11-cis-retinal synthesis) for rod and cone pathophysiology.

Expression of leukemia inhibitory factor in Müller glia cells is regulated by a redox-dependent mRNA stability mechanism.

BACKGROUND: Photoreceptor degeneration is a main hallmark of many blinding diseases making protection of photoreceptors crucial to prevent vision loss. Thus, regulation of endogenous neuroprotective factors may be key for cell survival and attenuation of disease progression. Important neuroprotective factors in the retina include H2O2 generated by injured photoreceptors, and leukemia inhibitory factor (LIF) expressed in Müller glia cells in response to photoreceptor damage. RESULTS: We present evidence that H2O2 connects to the LIF response by inducing stabilization of Lif transcripts in Müller cells. This process was independent of active gene transcription and p38 MAPK, but relied on AU-rich elements (AREs), which we identified within the highly conserved Lif 3'UTR. Affinity purification combined with quantitative mass spectrometry identified several proteins that bound to these AREs. Among those, interleukin enhancer binding factor 3 (ILF3) was confirmed to participate in the redox-dependent Lif mRNA stabilization. Additionally we show that KH-type splicing regulatory protein (KHSRP) was crucial for maintaining basal Lif expression levels in non-stressed Müller cells. CONCLUSIONS: Our results suggest that H2O2-induced redox signaling increases Lif transcript levels through ILF3 mediated mRNA stabilization. Generation of H2O2 by injured photoreceptors may thus enhance stability of Lif mRNA and therefore augment neuroprotective LIF signaling during degenerative conditions in vivo.

The erythropoietin receptor is not required for the development, function, and aging of rods and cells in the retinal periphery.

PURPOSE: Erythropoietin (EPO) was originally described for its antiapoptotic effects on erythroid progenitor cells in bone marrow. In recent years, however, EPO has also been shown to be cytoprotective in several tissues, including the retina. There, exogenous application of EPO was reported to exert neuro- and vasoprotection in several models of retinal injury. EPO and the erythropoietin receptor (EPOR) are expressed in the retina, but the role of endogenous EPO-EPOR signaling in this tissue remains elusive. Here, we investigated the consequences for cell physiology and survival when EpoR is ablated in rod photoreceptors or in the peripheral retina. METHODS: Two mouse lines were generated harboring a cyclization recombinase (CRE)-mediated knockdown of EpoR in rod photoreceptors (EpoR(flox/flox);Opn-Cre) or in a heterogeneous cell population of the retinal periphery (EpoR(flox/flox);α-Cre). The function of the retina was measured with electroretinography. Retinal morphology was analyzed in tissue sections. The vasculature of the retina was investigated on flatmount preparations, cryosections, and fluorescein angiography. Retinal nuclear layers were isolated by laser capture microdissection to test for EpoR expression. Gene expression analysis was performed with semiquantitative real-time PCR. To test if the absence of EPOR potentially increases retinal susceptibility to hypoxic stress, the knockdown mice were exposed to hypoxia. RESULTS: Newborn mice had lower retinal expression levels of EpoR and soluble EpoR (sEpoR) than the adult wild-type mice. In the adult mice, the EpoR transcripts were elevated in the inner retinal layers, while expression in the photoreceptors was low. CRE-mediated deletion in the EpoR(flox/flox);Opn-Cre mice led to a decrease in EpoR mRNA expression in the outer nuclear layer. A significant decrease in EpoR expression was measured in the retina of the EpoR(flox/flox);α-Cre mice, accompanied by a strong and significant decrease in sEpoR expression. Analysis of the retinal morphology in the two knockdown lines did not reveal any developmental defects or signs of accelerated degeneration in the senescent tissue. Similarly, retinal function was not altered under scotopic and photopic conditions. In addition, EpoR knockdown had no influence on cell viability under acute hypoxic conditions. Retinal angiogenesis and vasculature were normal in the absence of EPOR. However, expression of some EPOR-signaling target genes was significantly altered in the retinas of the EpoR(flox/flox);α-Cre mice. CONCLUSIONS: Our data suggest that expression of EPOR in rod photoreceptors, Müller cells, and amacrine, horizontal, and ganglion cells of the peripheral retina is not required for the maturation, function, and survival of these cells in aging tissue. Based on the expression of the EPOR-signaling target genes, we postulate that expression of soluble EPOR in the retina may modulate endogenous EPO-EPOR signaling.

Mouse models for cone degeneration.

Loss of cone vision has devastating effects on everyday life. Even though much effort has been made to understand cone physiology and pathophysiology, no successful therapies are available for patients suffering from cone disorders. As complex retinal interactions cannot be studied in vitro, utilization of different animal models is inevitable. Due to recent advances in transgenesis, mice became the most popular animal model to study human diseases, also in ophthalmology. While there are similarities in retinal anatomy and pathophysiology between mice and humans, there are also differences, most importantly the lack of a cone-rich macula in mice. Instead, cones in mice are rare and distributed over the whole retina, which makes the analysis of cone pathophysiology very difficult in these animals. This hindrance is one of the reasons why our understanding of rod pathophysiological processes is much more advanced. Recently, however, the sparseness of cones was overcome by the generation of the Nrl (- / -) mouse that expresses only cone photoreceptors in the retina. This paper will give a brief overview of some of the known mouse models to study cone degeneration and discuss the current knowledge gained from the analysis of these models.

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.

Activation of survival pathways in the degenerating retina of rd10 mice.

Blinding diseases of the retina are frequently characterized by loss of photoreceptor cells. The retinal degeneration 10 (rd10) mouse expresses a mutant form of rod phosphodiesterase leading to autosomal recessive photoreceptor degeneration. In contrast to rd1, rd10 mice have remaining rod function mimicking more closely most forms of human Retinitis Pigmentosa. Here we use morphology, biochemistry, retinal whole mounts, real-time PCR, Western blotting and immunofluorescence to compile a comprehensive report on progression of retinal degeneration in the rd10 retina up to one year of age. We show that retinal development, morphology, gene expression pattern and retinal vasculature was normal until postnatal day 15. Thereafter, a bi-phasic pattern of rod cell death emerged with a first rapid phase peaking around 3 weeks of age followed by a slower second phase. Death of cone cells followed with a delay and vessel dropout was prominent in the retinal periphery of 6 months old rd10 mice. At one year of age, RPE atrophy was evident. The degenerating retina rapidly induced expression of transcriptional regulators Atf3 and Cebpd. Induction of Atf3 was transient and lasted only for several days at the beginning of degeneration whereas levels of Cebpd remained elevated throughout the period of photoreceptor loss. Several protective genes such as Lif, Edn2 and Fgf2 which are implicated in a potent endogenous survival pathway, and Mt1 and Mt2 were strongly upregulated in the rd10 retina. In addition, increased expression of Casp1 and Il1b suggested an inflammatory response.