Diabetic Retinopathy: From Animal Models to Cellular Signaling.

Diabetic retinopathy (DR) is an ocular complication of diabetes mellitus (DM), a metabolic disorder characterized by elevation in blood glucose level. The pathogenesis of DR includes vascular, neuronal, and inflammatory components leading to activation of complex cellular molecular signaling. If untreated, the disease can culminate in vision loss that eventually leads to blindness. Animal models mimicking different aspects of DM complications have been developed to study the development and progression of DR. Despite the significant contribution of the developed DR models to discovering the mechanisms of DR and the recent achievements in the research field, the sequence of cellular events in diabetic retinas is still under investigation. Partially, this is due to the complexity of molecular mechanisms, although the lack of availability of models that adequately mimic all the neurovascular pathobiological features observed in patients has also contributed to the delay in determining a precise molecular trigger. In this review, we provide an update on the status of animal models of DR to help investigators choose an appropriate system to validate their hypothesis. We also discuss the key cellular and physiological events of DR in these models.

Neuronatin Protein in Health and Disease.

Neuronatin (NNAT) was first identified as a brain-specific gene crucial for brain development. Over the years, NNAT has been studied in different developing and post-developed tissues and organs. While NNAT manifests functional and structural similarities to the phospholamban gene, its physiological and pathological roles in healthy and diseased tissues have not been precisely identified. Ca2+ signaling, glucose transport, insulin secretion, and inflammation modulated at different pathological conditions have been proposed to be governed by NNAT. This review describes the current findings of cellular molecular pathways known to be modified concomitantly with an alteration in NNAT expression, and it highlights the need to conduct extensive investigation regarding the role of NNAT in health and disease. J. Cell. Physiol. 232: 477-481, 2017. © 2016 Wiley Periodicals, Inc.

Signalling beyond photon absorption: extracellular retinoids and growth factors modulate rod photoreceptor sensitivity.

KEY POINTS: We propose that the end product of chromophore bleaching in rod photoreceptors, all-trans retinol, is part of a feedback loop that increases the sensitivity of the phototransduction cascade in rods. A previously described light-induced hypersensitivity in rods, termed adaptive potentiation, is reduced by exogenously applied all-trans retinol but not all-trans retinal. This potentiation is produced by insulin-like growth factor-1, whose binding proteins are located in the extracellular matrix, even in our isolated retina preparation after removal of the retinal pigmented epithelium. Simple modelling suggests that the light stimuli used in the present study will produce sufficient all-trans retinol within the interphotoreceptor matrix to explain the potentiation effect. ABSTRACT: Photoreceptors translate the absorption of photons into electrical signals for propagation through the visual system. Mammalian photoreceptor signalling has largely been studied in isolated cells, and such studies have necessarily avoided the complex environment of supportive proteins that surround the photoreceptors. The interphotoreceptor matrix (IPM) contains an array of proteins that aid in both structural maintenance and cellular homeostasis, including chromophore turnover. In signalling photon absorption, the chromophore 11-cis retinal is first isomerized to all-trans retinal, followed by conversion to all-trans retinol (ROL) for removal from the photoreceptor. Interphotoreceptor retinoid-binding protein (IRBP) is the most abundant protein in the IPM, and it promotes the removal of bleached chromophores and recycling in the nearby retinal pigment epithelium. By studying the light responses of isolated mouse retinas, we demonstrate that ROL can act as a feedback signal onto photoreceptors that influences the sensitivity of phototransduction. In addition to IRBP, the IPM also contains insulin-like growth factor-1 (IGF-1) and its associated binding proteins, although their functions have not yet been described. We demonstrate that extracellular application of physiological concentrations of IGF-1 can increase rod photoreceptor sensitivity in mammalian retinas. We also determine that chromophores and growth factors can limit the range of a newly described form of photoreceptor light adaptation. Finally, fluorescent antibodies demonstrate the presence of IRBP and IGFBP-3 in isolated retinas. A simple model of the formation and release of ROL into the extracellular space quantitatively describes this novel feedback loop.

Selective vulnerability of the intermediate retinal capillary plexus precedes retinal ganglion cell loss in ocular hypertension.

Introduction: Glaucoma, a disease of retinal ganglion cell (RGC) injury and potentially devastating vision loss, is associated with both ocular hypertension (OHT) and reduced ocular blood flow. However, the relationship between OHT and retinal capillary architecture is not well understood. In this project, we studied microvasculature damage in mice exposed to mild levels of induced OHT. Methods: Mild OHT was induced with the microbead model for 2 weeks. At this time point, some retinas were immunostained with CD31 (endothelium), Collagen IV (basement membrane), and RBPMS (RGCs) for z-stack confocal microscopy. We processed these confocal images to distinguish the three retinal capillary plexi (superficial, intermediate, and deep). We manually counted RGC density, analyzed vascular complexity, and identified topographical and spatial vascular features of the retinal capillaries using a combination of novel manual and automated workflows. Other retinas were dissociated and immunopanned to isolate RGCs and amacrine cells (ACs) for hypoxia gene array analysis. Results: RGC counts were normal but there was decreased overall retinal capillary complexity. This reduced complexity could be explained by abnormalities in the intermediate retinal capillary plexus (IRCP) that spared the other plexi. Capillary junction density, vessel length, and vascular area were all significantly reduced, and the number of acellular capillaries was dramatically increased. ACs, which share a neurovascular unit (NVU) with the IRCP, displayed a marked increase in the relative expression of many hypoxia-related genes compared to RGCs from the same preparations. Discussion: We have discovered a rapidly occurring, IRCP-specific, OHT-induced vascular phenotype that precedes RGC loss. AC/IRCP NVU dysfunction may be a mechanistic link for early vascular remodeling in glaucoma.

A Novel Tree Shrew Model of Diabetic Retinopathy.

Existing animal models with rod-dominant retinas have shown that hyperglycemia injures neurons, but it is not yet clearly understood how blue cone photoreceptors and retinal ganglion cells (RGCs) deteriorate in patients because of compromised insulin tolerance. In contrast, northern tree shrews (Tupaia Belangeri), one of the closest living relatives of primates, have a cone-dominant retina with short wave sensitivity (SWS) and long wave sensitivity (LWS) cones. Therefore, we injected animals with a single streptozotocin dose (175 mg/kg i.p.) to investigate whether sustained hyperglycemia models the features of human diabetic retinopathy (DR). We used the photopic electroretinogram (ERG) to measure the amplitudes of A and B waves and the photopic negative responses (PhNR) to evaluate cone and RGC function. Retinal flat mounts were prepared for immunohistochemical analysis to count the numbers of neurons with antibodies against cone opsins and RGC specific BRN3a proteins. The levels of the proteins TRIB3, ISR-1, and p-AKT/p-mTOR were measured with western blot. The results demonstrated that tree shrews manifested sustained hyperglycemia leading to a slight but significant loss of SWS cones (12%) and RGCs (20%) 16 weeks after streptozotocin injection. The loss of BRN3a-positive RGCs was also reflected by a 30% decline in BRN3a protein expression. These were accompanied by reduced ERG amplitudes and PhNRs. Importantly, the diabetic retinas demonstrated increased expression of TRIB3 and level of p-AKT/p-mTOR axis but reduced level of IRS-1 protein. Therefore, a new non-primate model of DR with SWS cone and RGC dysfunction lays the foundation to better understand retinal pathophysiology at the molecular level and opens an avenue for improving the research on the treatment of human eye diseases.

Tribbles Homolog 3 Mediates the Development and Progression of Diabetic Retinopathy.

The current understanding of the molecular pathogenesis of diabetic retinopathy does not provide a mechanistic link between early molecular changes and the subsequent progression of the disease. In this study, we found that human diabetic retinas overexpressed TRIB3 and investigated the role of TRIB3 in diabetic retinal pathobiology in mice. We discovered that TRIB3 controlled major molecular events in early diabetic retinas via HIF1α-mediated regulation of retinal glucose flux, reprogramming cellular metabolism, and governing of inflammatory gene expression. These early molecular events further defined the development of neurovascular deficit observed in mice with diabetic retinopathy. TRIB3 ablation in the streptozotocin-induced mouse model led to significant retinal ganglion cell survival and functional restoration accompanied by a dramatic reduction in pericyte loss and acellular capillary formation. Under hypoxic conditions, TRIB3 contributed to advanced proliferative stages by significant upregulation of GFAP and VEGF expression, thus controlling gliosis and aberrant vascularization in oxygen-induced retinopathy mouse retinas. Overall, our data reveal that TRIB3 is a master regulator of diabetic retinal pathophysiology that may accelerate the onset and progression of diabetic retinopathy to proliferative stages in humans and present TRIB3 as a potentially novel therapeutic target for diabetic retinopathy.

Translational attenuation and retinal degeneration in mice with an active integrated stress response.

An integrated stress response (ISR), identified in several different animal models of inherited retinal degeneration (IRD), is activated following various cellular stresses. The ISR results in the phosphorylation of eIF2α (p-eIF2α) and a consequent halt in protein synthesis. Although generally protective, persistent elevations in p-eIF2α could lead to cell demise. Therefore, we aimed to determine whether ISR activation is associated with diminished translation rates in mice with IRD. Retinal protein extracts from rd16 mice at different time points were analyzed and the retinal levels of protein synthesis were assessed using the SUnSET method. We found that rd16 mice experience persistent ISR activation: p-eIF2α, ATF4, and CHOP were significantly upregulated at P15 and P20. In agreement with ISR activation, we found that rd16 mice experience translational attenuation at P15. Similar to rd16, other IRD models, T17M RHO, and rd10 also demonstrated a decline in protein synthesis, correlating with p-eIF2α elevation. We then assessed the role of PERK and eIF2α in translational attenuation in rd16 using a PERK inhibitor, GSK2606414. We found that while the treatment significantly reduced p-eIF2α, it did not cause a complete recovery in translation. This suggests that eIF2α is not the only or even the primary point of translational control in IRD, and a second node of translational regulation comprising AKT and mTOR should be evaluated. Surprisingly, we found that AKT-mTOR signaling was diminished in rd16 and rd10 retinas, suggesting a potential link between AKT-mTOR and translational inhibition. Therefore, for the first time, this study shows translation attenuation in IRD models, and highlights the potential roles of eIF2α kinases and AKT-mTOR signaling that could grant valuable insight into the potential treatments for IRD.

Neurodegeneration: Keeping ATF4 on a Tight Leash.

Activation of the endoplasmic reticulum (ER) stress and ER stress response, also known as the unfolded protein response (UPR), is common to various degenerative disorders. Therefore, signaling components of the UPR are currently emerging as potential targets for intervention and treatment of human diseases. One UPR signaling member, activating transcription factor 4 (ATF4), has been found up-regulated in many pathological conditions, pointing to therapeutic potential in targeting its expression. In cells, ATF4 governs multiple signaling pathways, including autophagy, oxidative stress, inflammation, and translation, suggesting a multifaceted role of ATF4 in the progression of various pathologies. However, ATF4 has been shown to trigger both pro-survival and pro-death pathways, and this, perhaps, can explain the contradictory opinions in current literature regarding targeting ATF4 for clinical application. In this review, we summarized recent published studies from our labs and others that focus on the therapeutic potential of the strategy controlling ATF4 expression in different retinal and neurodegenerative disorders.

Neuronatin is a stress-responsive protein of rod photoreceptors.

Neuronatin (NNAT) is a small transmembrane proteolipid that is highly expressed in the embryonic developing brain and several other peripheral tissues. This study is the first to provide evidence that NNAT is detected in the adult retina of various adult rod-dominant mammals, including wild-type (WT) rodents, transgenic rodents expressing mutant S334ter, P23H, or T17M rhodopsin, non-human primates, humans, and cone-dominant tree shrews. Immunohistochemical and quantitative real time polymerase chain reaction (qRT-PCR) analyses were applied to detect NNAT. Confocal microscopy analysis revealed that NNAT immunofluorescence is restricted to the outer segments (OSs) of photoreceptors without evidence of staining in other retinal cell types across all mammalian species. Moreover, in tree shrew retinas, we found NNAT to be co-localized with rhodopsin, indicating its predominant expression in rods. The rod-derived expression of NNAT was further confirmed by qRT-PCR in isolated rod photoreceptor cells. We also used these cells to mimic cellular stress in transgenic retinas by treating them with the endoplasmic reticulum stress inducer, tunicamycin. Thus, our data revealed accumulation of NNAT around the nucleus as compared to dispersed localization of NNAT within control cells. This distribution coincided with the partial intracellular mislocalization of NNAT to the outer nuclear layer observed in transgenic retinas. In addition, stressed retinas demonstrated an increase of NNAT mRNA and protein levels. Therefore, our study demonstrated that NNAT is a novel stress-responsive protein with a potential structural and/or functional role in adult mammalian retinas.