The stress response protein REDD1 promotes diabetes-induced oxidative stress in the retina by Keap1-independent Nrf2 degradation.

The transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2) plays a critical role in reducing oxidative stress by promoting the expression of antioxidant genes. Both individuals with diabetes and preclinical diabetes models exhibit evidence of a defect in retinal Nrf2 activation. We recently demonstrated that increased expression of the stress response protein regulated in development and DNA damage 1 (REDD1) is necessary for the development of oxidative stress in the retina of streptozotocin-induced diabetic mice. In the present study, we tested the hypothesis that REDD1 suppresses the retinal antioxidant response to diabetes by repressing Nrf2 function. We found that REDD1 ablation enhances Nrf2 DNA-binding activity in the retina and that the suppressive effect of diabetes on Nrf2 activity is absent in the retina of REDD1-deficient mice compared with WT. In human MIO-M1 Müller cell cultures, REDD1 deletion prevented oxidative stress in response to hyperglycemic conditions, and this protective effect required Nrf2. REDD1 suppressed Nrf2 stability by promoting its proteasomal degradation independently of Nrf2's interaction with Kelch-like ECH-associated protein 1 (Keap1), but REDD1-mediated Nrf2 degradation required glycogen synthase kinase 3 (GSK3) activity and Ser-351/Ser-356 of Nrf2. Diabetes diminished inhibitory phosphorylation of glycogen synthase kinase 3ß (GSK3ß) at Ser-9 in the retina of WT mice but not in REDD1-deficient mice. Pharmacological inhibition of GSK3 enhanced Nrf2 activity and prevented oxidative stress in the retina of diabetic mice. The findings support a model wherein hyperglycemia-induced REDD1 blunts the Nrf2 antioxidant response to diabetes by activating GSK3, which, in turn, phosphorylates Nrf2 to promote its degradation.

Descriptive analysis of Fibulin-3 and the extracellular vesicle marker, Alix, in drusen from a small cohort of postmortem human eyes.

Fibulin-3 (Fib3) is a secreted glycoprotein that is expressed in the retina and has been associated with drusen formation in age-related macular degeneration (AMD). The purpose of this study was to assess whether Fib3 is associated with extracellular vesicles (EVs) in drusen from non-diseased and AMD human donors. De-identified sections of human eyes were received from the National Disease Research Institute (NDRI, Philadelphia). Donor eyes were either non-diseased (no known ocular pathology) or had been diagnosed with AMD. Retinal cryostat sections were labeled with primary antibodies targeted to Fib3, Apolipoprotein E (ApoE; a drusen marker), and ALG-2 interacting protein X (Alix, an EV marker) for confocal imaging (Leica TCS SP8). Fib3-positive (Fib3+) puncta were detected on the apical region of the RPE layer and within large AMD drusen. Alix-positive (Alix+) puncta were also detected in a single AMD druse, where a number were Fib3+ and the remaining were Fib3-negative. Similarly, there were Fib3+ puncta that were Alix-negative. Fib3 and Alix also showed a degree of colocalization in the photoreceptor outer segments of the neural retina. Our data suggest that the Alix+ puncta are EV-rich populations that accumulate, together with Fib3, within the drusen matrix during AMD. The EV population is likely heterogeneous, such that there are sub-populations with different cargo content.

Nrf2 as molecular target for polyphenols: A novel therapeutic strategy in diabetic retinopathy.

Diabetic retinopathy is a microvascular complication of diabetes that is considered one of the leading causes of blindness among adults. More than 4.4 million people suffer from this disorder throughout the world. Growing evidence suggests that oxidative stress plays a crucial role in the pathophysiology of diabetic retinopathy. Nuclear factor erythroid 2-related factor 2 (Nrf2), a redox sensitive transcription factor, plays an essential protective role in regulating the physiological response to oxidative and electrophilic stress via regulation of multiple genes encoding antioxidant proteins and phase II detoxifying enzymes. Many studies suggest that dozens of natural compounds, including polyphenols, can supress oxidative stress and inflammation through targeting Nrf2 and consequently activating the antioxidant response element-related cytoprotective genes. Therefore, Nrf2 may provide a new therapeutic target for treatment of diabetic retinopathy. In the present article, we will focus on the role of Nrf2 in diabetic retinopathy and the ability of polyphenols to target Nrf2 as a therapeutic strategy.

NRF2 plays a protective role in diabetic retinopathy in mice.

AIMS/HYPOTHESIS: Although much is known about the pathophysiological processes contributing to diabetic retinopathy (DR), the role of protective pathways has received less attention. The transcription factor nuclear factor erythroid-2-related factor 2 (also known as NFE2L2 or NRF2) is an important regulator of oxidative stress and also has anti-inflammatory effects. The objective of this study was to explore the potential role of NRF2 as a protective mechanism in DR. METHODS: Retinal expression of NRF2 was investigated in human donor and mouse eyes by immunohistochemistry. The effect of NRF2 modulation on oxidative stress was studied in the human Müller cell line MIO-M1. Non-diabetic and streptozotocin-induced diabetic wild-type and Nrf2 knockout mice were evaluated for multiple DR endpoints. RESULTS: NRF2 was expressed prominently in Müller glial cells and astrocytes in both human and mouse retinas. In cultured MIO-M1 cells, NRF2 inhibition significantly decreased antioxidant gene expression and exacerbated tert-butyl hydroperoxide- and hydrogen peroxide-induced oxidative stress. NRF2 activation strongly increased NRF2 target gene expression and suppressed oxidant-induced reactive oxygen species. Diabetic mice exhibited retinal NRF2 activation, indicated by nuclear translocation. Superoxide levels were significantly increased by diabetes in Nrf2 knockout mice as compared with wild-type mice. Diabetic Nrf2 knockout mice exhibited a reduction in retinal glutathione and an increase in TNF-α protein compared with wild-type mice. Nrf2 knockout mice exhibited early onset of blood-retina barrier dysfunction and exacerbation of neuronal dysfunction in diabetes. CONCLUSIONS/INTERPRETATION: These results indicate that NRF2 is an important protective factor regulating the progression of DR and suggest enhancement of the NRF2 pathway as a potential therapeutic strategy.

NLRP3 Inflammasome Priming in the Retina of Diabetic Mice Requires REDD1-Dependent Activation of GSK3ß.

Purpose: Inflammasome activation has been implicated in the development of retinal complications caused by diabetes. This study was designed to identify signaling events that promote retinal NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation in response to diabetes. Methods: Diabetes was induced in mice by streptozotocin administration. Retinas were examined after 16 weeks of diabetes. Human MIO-M1 Müller cells were exposed to hyperglycemic culture conditions. Genetic and pharmacological interventions were used to interrogate signaling pathways. Visual function was assessed in mice using a virtual optomotor system. Results: In the retina of diabetic mice and in Müller cell cultures, NLRP3 and interleukin-1ß (IL-1ß) were increased in response to hyperglycemic conditions and the stress response protein Regulated in Development and DNA damage 1 (REDD1) was required for the effect. REDD1 deletion prevented caspase-1 activation in Müller cells exposed to hyperglycemic conditions and reduced IL-1ß release. REDD1 promoted nuclear factor κB signaling in cells exposed to hyperglycemic conditions, which was necessary for an increase in NLRP3. Expression of a constitutively active GSK3ß variant restored NLRP3 expression in REDD1-deficient cells exposed to hyperglycemic conditions. GSK3 activity was necessary for increased NLRP3 expression in the retina of diabetic mice and in cells exposed to hyperglycemic conditions. Müller glia-specific REDD1 deletion prevented increased retinal NLRP3 levels and deficits in contrast sensitivity in diabetic mice. Conclusions: The data support a role for REDD1-dependent activation of GSK3ß in NLRP3 inflammasome transcriptional priming and in the production of IL-1ß by Müller glia in response to diabetes.

Minocycline prevents retinal inflammation and vascular permeability following ischemia-reperfusion injury.

BACKGROUND: Many retinal diseases are associated with vascular dysfunction accompanied by neuroinflammation. We examined the ability of minocycline (Mino), a tetracycline derivative with anti-inflammatory and neuroprotective properties, to prevent vascular permeability and inflammation following retinal ischemia-reperfusion (IR) injury, a model of retinal neurodegeneration with breakdown of the blood-retinal barrier (BRB). METHODS: Male Sprague-Dawley rats were subjected to 45 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Rats were treated with Mino prior to and following IR. At 48 h after reperfusion, retinal gene expression, cellular inflammation, Evan's blue dye leakage, tight junction protein organization, caspase-3 activation, and DNA fragmentation were measured. Cellular inflammation was quantified by flow-cytometric evaluation of retinal tissue using the myeloid marker CD11b and leukocyte common antigen CD45 to differentiate and quantify CD11b+/CD45low microglia, CD11b+/CD45hi myeloid leukocytes and CD11bneg/CD45hi lymphocytes. Major histocompatibility complex class II (MHCII) immunoreactivity was used to determine the inflammatory state of these cells. RESULTS: Mino treatment significantly inhibited IR-induced retinal vascular permeability and disruption of tight junction organization. Retinal IR injury significantly altered mRNA expression for 21 of 25 inflammation- and gliosis-related genes examined. Of these, Mino treatment effectively attenuated IR-induced expression of lipocalin 2 (LCN2), serpin peptidase inhibitor clade A member 3 N (SERPINA3N), TNF receptor superfamily member 12A (TNFRSF12A), monocyte chemoattractant-1 (MCP-1, CCL2) and intercellular adhesion molecule-1 (ICAM-1). A marked increase in leukostasis of both myeloid leukocytes and lymphocytes was observed following IR. Mino treatment significantly reduced retinal leukocyte numbers following IR and was particularly effective in decreasing the appearance of MHCII+ inflammatory leukocytes. Surprisingly, Mino did not significantly inhibit retinal cell death in this model. CONCLUSIONS: IR induces a retinal neuroinflammation within hours of reperfusion characterized by inflammatory gene expression, leukocyte adhesion and invasion, and vascular permeability. Despite Mino significantly inhibiting these responses, it failed to block neurodegeneration.

Synthesis and structure-activity relationships of 2-amino-3-carboxy-4-phenylthiophenes as novel atypical protein kinase C inhibitors.

Recent evidence suggests atypical protein kinase C (aPKC) isoforms are required for both TNF- and VEGF-induced breakdown of the blood-retinal barrier (BRB) and endothelial permeability to 70kDa dextran or albumin. A chemical library screen revealed a series of novel small molecule phenylthiophene based inhibitors of aPKC isoforms that effectively block permeability in cell culture and in vivo. In an effort to further elucidate the structural requirements of this series of inhibitors, we detail in this study a structure-activity relationship (SAR) built on screening hit 1, which expands on our initial pharmacophore model. The biological activity of our analogues was evaluated in models of bona fide aPKC-dependent signaling including NFκB driven-gene transcription as a marker for an inflammatory response and VEGF/TNF-induced vascular endothelial permeability. The EC50 for the most efficacious inhibitors (6, 32) was in the low nanomolar range in these two cellular assays. Our study demonstrates the key structural elements that confer inhibitory activity and highlights the requirement for electron-donating moieties off the C-4 aryl moiety of the 2-amino-3-carboxy-4-phenylthiophene backbone. These studies suggest that this class has potential for further development into small molecule aPKC inhibitors with therapeutic efficacy in a host of diseases involving increased vascular permeability and inflammation.

Extracellular vesicles from retinal pigment epithelial cells expressing R345W-Fibulin-3 induce epithelial-mesenchymal transition in recipient cells.

We have shown previously that expression of R345W-Fibulin-3 induces epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. The purpose of the current study was to determine if extracellular vesicles (EVs) derived from RPE cells expressing R345W-Fibulin-3 mutation are sufficient to induce EMT in recipient cells. ARPE-19 cells were infected with luciferase-tagged wild-type (WT)- Fibulin-3 or luciferase-tagged R345W-Fibulin-3 (R345W) using lentiviruses. EVs were isolated from the media by ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy and cryogenic electron microscopy were performed to study the morphology of the EVs. The size distribution of EVs were determined by nanoparticle tracking analysis (NTA). EV cargo was analysed using LC-MS/MS based proteomics. EV-associated transforming growth factor beta 1 (TGFß1) protein was measured by enzyme-linked immunosorbent assay. The capacity of EVs to stimulate RPE migration was evaluated by treating recipient cells with WT- or R345W-EVs. The role of EV-bound TGFß was determined by pre-incubation of EVs with a pan-TGFß blocking antibody or IgG control. EM imaging revealed spherical vesicles with two subpopulations of EVs: a group with diameters around 30 nm and a group with diameters over 100 nm, confirmed by NTA analysis. Pathway analysis revealed that members of the sonic hedgehog pathway were less abundant in R345W- EVs, while EMT drivers were enriched. Additionally, R345W-EVs had higher concentrations of TGFß1 compared to control. Critically, treatment with R345W-EVs was sufficient to increase EMT marker expression, as well as cell migration in recipient cells. This EV-increased cell migration was significantly inhibited by pre-incubation of EVs with pan-TGFß-neutralising antibody. In conclusion, the expression of R345W-Fibulin-3 alters the size and cargo of EVs, which are sufficient to enhance the rate of cell migration in a TGFß dependent manner. These results suggest that EV-bound TGFß plays a critical role in the induction of EMT in RPE cells.

Müller Glial Expression of REDD1 Is Required for Retinal Neurodegeneration and Visual Dysfunction in Diabetic Mice.

Clinical studies support a role for the protein regulated in development and DNA damage response 1 (REDD1) in ischemic retinal complications. To better understand how REDD1 contributes to retinal pathology, we examined human single-cell sequencing data sets and found specificity of REDD1 expression that was consistent with markers of retinal Müller glia. Thus, we investigated the hypothesis that REDD1 expression specifically in Müller glia contributes to diabetes-induced retinal pathology. The retina of Müller glia-specific REDD1 knockout (REDD1-mgKO) mice exhibited dramatic attenuation of REDD1 transcript and protein expression. In the retina of streptozotocin-induced diabetic control mice, REDD1 protein expression was enhanced coincident with an increase in oxidative stress. In the retina of diabetic REDD1-mgKO mice, there was no increase in REDD1 protein expression, and oxidative stress was reduced compared with diabetic control mice. In both Müller glia within the retina of diabetic mice and human Müller cell cultures exposed to hyperglycemic conditions, REDD1 was necessary for increased expression of the gliosis marker glial fibrillary acidic protein. The effect of REDD1 deletion in preventing gliosis was associated with suppression of oxidative stress and required the antioxidant transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2). In contrast to diabetic control mice, diabetic REDD1-mgKO mice did not exhibit retinal thinning, increased markers of neurodegeneration within the retinal ganglion cell layer, or deficits in visual function. Overall, the findings support a key role for Müller glial REDD1 in the failed adaptive response of the retina to diabetes that includes gliosis, neurodegeneration, and impaired vision.

Neurodegeneration, Neuroprotection and Regeneration in the Zebrafish Retina.

Neurodegenerative retinal diseases, such as glaucoma and diabetic retinopathy, involve a gradual loss of neurons in the retina as the disease progresses. Central nervous system neurons are not able to regenerate in mammals, therefore, an often sought after course of treatment for neuronal loss follows a neuroprotective or regenerative strategy. Neuroprotection is the process of preserving the structure and function of the neurons that have survived a harmful insult; while regenerative approaches aim to replace or rewire the neurons and synaptic connections that were lost, or induce regrowth of damaged axons or dendrites. In order to test the neuroprotective effectiveness or the regenerative capacity of a particular agent, a robust experimental model of retinal neuronal damage is essential. Zebrafish are being used more often in this type of study because their eye structure and development is well-conserved between zebrafish and mammals. Zebrafish are robust genetic tools and are relatively inexpensive to maintain. The large array of functional and behavioral tests available in zebrafish makes them an attractive model for neuroprotection studies. Some common insults used to model retinal disease and study neuroprotection in zebrafish include intense light, chemical toxicity and mechanical damage. This review covers the existing retinal neuroprotection and regeneration literature in the zebrafish and highlights their potential for future studies.

Visual dysfunction associated with diabetic retinopathy.

Diabetic retinopathy is a leading cause of blindness and is commonly viewed as a vascular complication of diabetes mellitus. However, diabetes mellitus causes visual dysfunction before the onset of clinically visible microvascular changes associated with diabetic retinopathy. Thus, viewing diabetic retinopathy more generally as a neurovascular disease may lead to an improved understanding of the mechanisms responsible for vision loss. This article reviews the impact of diabetes mellitus on inner and outer retinal visual and electrophysiologic function and advocates for a multimodal approach to the study of diabetic retinopathy.

Angiotensin-(1-7) Attenuates Protein O-GlcNAcylation in the Retina by EPAC/Rap1-Dependent Inhibition of O-GlcNAc Transferase.

Purpose: O-GlcNAcylation of cellular proteins contributes to the pathophysiology of diabetes and evidence supports a role for augmented O-GlcNAcylation in diabetic retinopathy. The aim of this study was to investigate the impact of the renin-angiotensin system on retinal protein O-GlcNAcylation. Methods: Mice fed a high-fat diet were treated chronically with the angiotensin-converting enzyme inhibitor captopril or captopril plus the angiotensin-(1-7) Mas receptor antagonist A779. Western blotting and quantitative polymerase chain reaction were used to analyze retinal homogenates. Similar analyses were performed on lysates from human MIO-M1 retinal Müller cell cultures exposed to media supplemented with angiotensin-(1-7). Culture conditions were manipulated to influence the hexosamine biosynthetic pathway and/or signaling downstream of the Mas receptor. Results: In the retina of mice fed a high-fat diet, captopril attenuated protein O-GlcNAcylation in a manner dependent on Mas receptor activation. In MIO-M1 cells, angiotensin-(1-7) or adenylate cyclase activation were sufficient to enhance cyclic AMP (cAMP) levels and inhibit O-GlcNAcylation. The repressive effect of cAMP on O-GlcNAcylation was dependent on exchange protein activated by cAMP (EPAC), but not protein kinase A, and was recapitulated by a constitutively active variant of the small GTPase Rap1. We provide evidence that cAMP and angiotensin-(1-7) act to suppress O-GlcNAcylation by inhibition of O-GlcNAc transferase (OGT) activity. In cells exposed to an O-GlcNAcase inhibitor or hyperglycemic culture conditions, mitochondrial superoxide levels were elevated; however, angiotensin-(1-7) signaling prevented the effect. Conclusions: Angiotensin-(1-7) inhibits retinal protein O-GlcNAcylation via an EPAC/Rap1/OGT signaling axis.

Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells.

PURPOSE: To investigate the role of protein misfolding in retinal pigment epithelial (RPE) cell dysfunction, the effects of R345W-Fibulin-3 expression on RPE cell phenotype were studied. METHODS: Primary RPE cells were cultured to confluence on Transwells and infected with lentivirus constructs to express wild-type (WT)- or R345W-Fibulin-3. Barrier function was assessed by evaluating zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER). Polarized secretion of vascular endothelial growth factor (VEGF), was measured by Enzyme-linked immunosorbent assay (ELISA). Differentiation status was assessed by qPCR of genes known to be preferentially expressed in terminally differentiated RPE cells, and conversion to an epithelial-mesenchymal transition (EMT) phenotype was assessed by a migration assay. RESULTS: Compared to RPE cells expressing WT-Fibulin-3, ZO-1 distribution was disrupted and TER values were significantly lower in RPE cells expressing R345W-Fibulin-3. In cells expressing mutant Fibulin-3, VEGF secretion was attenuated basally but not in the apical direction, whereas Fibulin-3 secretion was reduced in both the apical and basal directions. Retinal pigment epithelial signature genes were downregulated and multiple genes associated with EMT were upregulated in the mutant group. Migration assays revealed a faster recovery rate in ARPE-19 cells overexpressing R345W-Fibulin-3 compared to WT. CONCLUSIONS: The results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells.

REDD1 Activates a ROS-Generating Feedback Loop in the Retina of Diabetic Mice.

Purpose: The present study was designed to evaluate the role of the stress response protein REDD1 in diabetes-induced oxidative stress and retinal pathology. Methods: Wild-type and REDD1-deficient mice were administered streptozotocin to induce diabetes. Some mice received the antioxidant N-acetyl-l-cysteine (NAC). Visual function was assessed by virtual optometry. Retinas were analyzed by Western blotting. Reactive oxygen species (ROS) were assessed by 2,7-dichlorofluoroscein. Similar analyses were performed on R28 retinal cells in culture exposed to hyperglycemic conditions, NAC, and/or the exogenous ROS source hydrogen peroxide. Results: In the retina of diabetic mice, REDD1 expression and ROS were increased. In cells in culture, hyperglycemic conditions enhanced REDD1 expression, ROS levels, and the mitochondrial membrane potential. However, similar effects were not observed in the retina of diabetic mice or cells lacking REDD1. In the retina of diabetic mice and cells exposed to hyperglycemic conditions, NAC normalized ROS and prevented an increase in REDD1 expression. Diabetic mice receiving NAC also exhibited improved contrast sensitivity as compared to diabetic controls. Hydrogen peroxide addition to culture medium increased REDD1 expression and attenuated Akt/GSK3 phosphorylation in a REDD1-dependent manner. In REDD1-deficient cells exposed to hyperglycemic conditions, expression of a dominant negative Akt or constitutively active GSK3 increased the mitochondrial membrane potential and promoted ROS. Conclusions: The findings provide new insight into the mechanism whereby diabetes-induced hyperglycemia causes oxidative stress and visual dysfunction. Specifically, hyperglycemia-induced REDD1 activates a ROS-generating feedback loop that includes Akt/GSK3. Thus, therapeutic approaches targeting REDD1 expression and ROS may be beneficial for preventing diabetes-induced visual dysfunction.

Retinal ganglion cells in diabetes.

Diabetic retinopathy has long been recognized as a vascular disease that develops in most patients, and it was believed that the visual dysfunction that develops in some diabetics was due to the vascular lesions used to characterize the disease. It is becoming increasingly clear that neuronal cells of the retina also are affected by diabetes, resulting in dysfunction and even degeneration of some neuronal cells. Retinal ganglion cells (RGCs) are the best studied of the retinal neurons with respect to the effect of diabetes. Although investigations are providing new information about RGCs in diabetes, including therapies to inhibit the neurodegeneration, critical information about the function, anatomy and response properties of these cells is yet needed to understand the relationship between RGC changes and visual dysfunction in diabetes.

Diabetes downregulates presynaptic proteins and reduces basal synapsin I phosphorylation in rat retina.

Diabetic retinopathy can result in vision loss and involves progressive neurovascular degeneration of the retina. This study tested the hypothesis that diabetes decreases the retinal expression of presynaptic proteins involved in synaptic function. The protein and mRNA contents for synapsin I, synaptophysin, vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa and postsynaptic density protein of 95 kDa were measured by immunohistochemistry, immunoblotting and real-time quantitative polymerase chain reaction in whole retinas and retinal synaptosomes from streptozotocin-diabetic and control Sprague-Dawley rats. There was less presynaptic protein immunoreactivity after 1 and 3 months of diabetes than in controls. Discrete synaptophysin-immunoreactive puncta were significantly smaller and fewer in sections from 1- and 3-month diabetic rat retinas than in those from controls. The content of presynaptic proteins was significantly less in whole retinas of 1- and 3-month diabetic rats, and in synaptosomes from 1-month diabetic rats, than in controls. Whole retinas had significantly less mRNA for these genes after 3 months but not 1 month of diabetes, as compared to controls (with the exception of postsynaptic density protein of 95 kDa). In contrast, there was significantly less mRNA for synaptic proteins in synaptosomes of 1-month diabetic rats than in controls, suggesting a localized depletion at synapses. Protein and mRNA for beta-actin and neuron-specific enolase were unchanged by diabetes. The ratio of phosphorylated to total synapsin I was also reduced in whole retina and isolated synaptosomes from 1-month diabetic rats, as compared to controls. These data suggest that diabetes has a profound impact on presynaptic protein expression in the retina, and may provide a mechanism for the well-established defects in vision and the electrophysiological response of the retina in diabetes.

Dendrite remodeling and other abnormalities in the retinal ganglion cells of Ins2 Akita diabetic mice.

PURPOSE: To determine the extent of retinal ganglion cell loss and morphologic abnormalities in surviving ganglion cells in Ins2 Akita/+ diabetic mice. METHODS: Mice that expressed cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) reporter genes under the transcriptional control of the Thy1 promoter were crossed with Ins2 Akita/+ mice. After 3 months of diabetes, the number and morphology of retinal ganglion cells was analyzed by confocal microscopy. The number of CFP-positive retinal ganglion cells was quantified in retinas of Ins2(Akita/+) Thy1-CFP mice. The morphology of surviving cells was examined, and dendritic density was quantified in Ins2 Akita/+ Thy1-YFP mice by using the Sholl analysis. RESULTS: Thy1-CFP expression was limited to retinal ganglion cell bodies. There was a 16.4% reduction in the density of CFP-positive ganglion cells in the peripheral retina of Ins2 Akita/+ mice compared with wild-type control retinas (P < 0.017), but no significant change in the central retina. Thy1-YFP expression occurred throughout the entire structure of a smaller number of cells, including their soma, axons, and dendrites. Six different morphologic clusters of cells were identified in the mouse retinas. The structure of dendrites of ON-type retinal ganglion cells was affected by diabetes, having 32.4% more dendritic terminals (P < 0.05), 18.6% increase in total dendrite length (P < 0.05), and 15.3% greater dendritic density compared with control retinas, measured by Scholl analysis. Abnormal swelling on somas, axons, and dendrites were noted in all subtypes of ganglion cells including those expressing melanopsin. CONCLUSIONS: The data show that retinal ganglion cells are lost from the peripheral retina of mice within the first 3 months of diabetes and that the dendrites of surviving large ON-type cells undergo morphologic changes. These abnormalities may explain some of the early anomalies in visual function induced by diabetes.

Short-Term Administration of Astaxanthin Attenuates Retinal Changes in Diet-Induced Diabetic Psammomys obesus.

OBJECTIVES: Psammomys obesus is a high-fat diet (HFD)-fed animal model of obesity and type 2 diabetes recently explored as a model of non-proliferative diabetic retinopathy. This study tested the protective effect of the pigment astaxanthin (AST) in the P. obesus diabetic retina. METHODS: Young adult P. obesus were randomly assigned to two groups. The control group received a normal diet consisting of a plant-based regimen, and the HFD group received an enriched laboratory chow. After 3 months, control and diabetic rodents were administered vehicle or AST, daily for 7 days. Body weight, blood glucose, and plasma pentosidine were assessed. Frozen sections of retinas were immunolabeled for markers of oxidative stress, glial reactivity and retinal ganglion cell bodies, and imaged by confocal microscopy. RESULTS: Retinal tissue from AST-treated control and HFD-diabetic P. obesus showed a greater expression of the antioxidant enzyme heme oxygenase-1 (HO-1). In retinas of HFD-diabetic AST-treated P. obesus, cellular retinaldehyde binding protein and glutamine synthetase in Müller cells were more intense compared to the untreated HFD-diabetic group. HFD-induced diabetes downregulated the expression of glial fibrillary acidic protein in astrocytes, the POU domain protein 3A in retinal ganglion cells, and synaptophysin throughout the plexiform layers. DISCUSSION: Our results show that type 2-like diabetes induced by HFD affected glial and neuronal retinal cell homeostasis. AST treatment induced the antioxidant enzyme HO-1 and reduced glial reactivity. These findings suggest that diabetic P. obesus is a useful model of HFD-induced obesity and diabetes to evaluate early neuroglial retinal alterations and antioxidant neuroprotection mechanisms in DR.

Deletion of the Akt/mTORC1 Repressor REDD1 Prevents Visual Dysfunction in a Rodent Model of Type 1 Diabetes.

Diabetes-induced visual dysfunction is associated with significant neuroretinal cell death. The current study was designed to investigate the role of the Protein Regulated in Development and DNA Damage Response 1 (REDD1) in diabetes-induced retinal cell death and visual dysfunction. We recently demonstrated that REDD1 protein expression was elevated in response to hyperglycemia in the retina of diabetic rodents. REDD1 is an important regulator of Akt and mammalian target of rapamycin and as such plays a key role in neuronal function and survival. In R28 retinal cells in culture, hyperglycemic conditions enhanced REDD1 protein expression concomitant with caspase activation and cell death. By contrast, in REDD1-deficient R28 cells, neither hyperglycemic conditions nor the absence of insulin in culture medium were sufficient to promote cell death. In the retinas of streptozotocin-induced diabetic mice, retinal apoptosis was dramatically elevated compared with nondiabetic controls, whereas no difference was observed in diabetic and nondiabetic REDD1-deficient mice. Electroretinogram abnormalities observed in b-wave and oscillatory potentials of diabetic wild-type mice were also absent in REDD1-deficient mice. Moreover, diabetic wild-type mice exhibited functional deficiencies in visual acuity and contrast sensitivity, whereas diabetic REDD1-deficient mice had no visual dysfunction. The results support a role for REDD1 in diabetes-induced retinal neurodegeneration.

Proteomic Analysis of Early Diabetic Retinopathy Reveals Mediators of Neurodegenerative Brain Diseases.

Purpose: Current evidence suggests that retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy. Our main goal was to examine whether, in the diabetic human retina, common proteins and pathways are shared with brain neurodegenerative diseases. Methods: A proteomic analysis was performed on three groups of postmortem retinas matched by age: nondiabetic control retinas (n = 5), diabetic retinas without glial activation (n = 5), and diabetic retinas with glial activation (n = 5). Retinal lysates from each group were pooled and run on an SDS-PAGE gel. Bands were analyzed sequentially by liquid chromatography-mass spectrometry (LC/MS) using an Orbitrap Mass Spectrometer. Results: A total of 2190 proteins were identified across all groups. To evaluate the association of the identified proteins with neurological signaling, significant signaling pathways belonging to the category "Neurotransmitters and Other Nervous System Signaling" were selected for analysis. Pathway analysis revealed that "Neuroprotective Role of THOP1 in Alzheimer's Disease" and "Unfolded Protein Response" pathways were uniquely enriched in control retinas. By contrast, "Dopamine Degradation" and "Parkinson's Signaling" were enriched only in diabetic retinas with glial activation. The "Neuregulin Signaling," "Synaptic Long Term Potentiation," and "Amyloid Processing" pathways were enriched in diabetic retinas with no glial activation. Conclusions: Diabetes-induced retinal neurodegeneration and brain neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, share common pathogenic pathways. These findings suggest that the study of neurodegeneration in the diabetic retina could be useful to further understand the neurodegenerative processes that occur in the brain of persons with diabetes.