Effects of Nox4 upregulation on PECAM-1 expression in a mouse model of diabetic retinopathy.

Diabetic Retinopathy (DR) is the leading cause of vision loss in working-age adults. The hallmark features of DR include vascular leakage, capillary loss, retinal ischemia, and aberrant neovascularization. Although the pathophysiology is not fully understood, accumulating evidence supports elevated reactive oxygen species associated with increased activity of NADPH oxidase 4 (Nox4) as major drivers of disease progression. Previously, we have shown that Nox4 upregulation in retinal endothelial cells by diabetes leads to increased vascular leakage by an unknown mechanism. Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell surface molecule that is highly expressed in endothelial cells and regulates endothelial barrier function. In the present study, using endothelial cell-specific human Nox4 transgenic (TG) mice and endothelial cell-specific Nox4 conditional knockout (cKO) mice, we investigated the impact of Nox4 upregulation on PECAM-1 expression in mouse retinas and brain microvascular endothelial cells (BMECs). Additionally, cultured human retinal endothelial cells (HRECs) transduced with adenovirus overexpressing human Nox4 were used in the study. We found that overexpression of Nox4 increases PECAM-1 mRNA but has no effect on its protein expression in the mouse retina, BMECs, or HRECs. Furthermore, PECAM-1 mRNA and protein expression was unchanged in BMECs isolated from cKO mice compared to wild type (WT) mice with or without 2 months of diabetes. Together, these findings do not support a significant role of Nox4 in the regulation of PECAM-1 expression in the diabetic retina and endothelial cells. Further studies are warranted to elucidate the mechanism of Nox4-induced vascular leakage by investigating other intercellular junctional proteins in endothelial cells and their implications in the pathophysiology of diabetic retinopathy.

Quantifying nanoscopic alterations associated with mitochondrial dysfunction using three-dimensional single-molecule localization microscopy.

Mitochondrial morphology provides unique insights into their integrity and function. Among fluorescence microscopy techniques, 3D super-resolution microscopy uniquely enables the analysis of mitochondrial morphological features individually. However, there is a lack of tools to extract morphological parameters from super-resolution images of mitochondria. We report a quantitative method to extract mitochondrial morphological metrics, including volume, aspect ratio, and local protein density, from 3D single-molecule localization microscopy images, with single-mitochondrion sensitivity. We validated our approach using simulated ground-truth SMLM images of mitochondria. We further tested our morphological analysis on mitochondria that have been altered functionally and morphologically in controlled manners. This work sets the stage to quantitatively analyze mitochondrial morphological alterations associated with disease progression on an individual basis.

The endoplasmic reticulum: Homeostasis and crosstalk in retinal health and disease.

The endoplasmic reticulum (ER) is the largest intracellular organelle carrying out a broad range of important cellular functions including protein biosynthesis, folding, and trafficking, lipid and sterol biosynthesis, carbohydrate metabolism, and calcium storage and gated release. In addition, the ER makes close contact with multiple intracellular organelles such as mitochondria and the plasma membrane to actively regulate the biogenesis, remodeling, and function of these organelles. Therefore, maintaining a homeostatic and functional ER is critical for the survival and function of cells. This vital process is implemented through well-orchestrated signaling pathways of the unfolded protein response (UPR). The UPR is activated when misfolded or unfolded proteins accumulate in the ER, a condition known as ER stress, and functions to restore ER homeostasis thus promoting cell survival. However, prolonged activation or dysregulation of the UPR can lead to cell death and other detrimental events such as inflammation and oxidative stress; these processes are implicated in the pathogenesis of many human diseases including retinal disorders. In this review manuscript, we discuss the unique features of the ER and ER stress signaling in the retina and retinal neurons and describe recent advances in the research to uncover the role of ER stress signaling in neurodegenerative retinal diseases including age-related macular degeneration, inherited retinal degeneration, achromatopsia and cone diseases, and diabetic retinopathy. In some chapters, we highlight the complex interactions between the ER and other intracellular organelles focusing on mitochondria and illustrate how ER stress signaling regulates common cellular stress pathways such as autophagy. We also touch upon the integrated stress response in retinal degeneration and diabetic retinopathy. Finally, we provide an update on the current development of pharmacological agents targeting the UPR response and discuss some unresolved questions and knowledge gaps to be addressed by future research.

Patterns of Gene Expression, Splicing, and Allele-Specific Expression Vary among Macular Tissues and Clinical Stages of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness, and elucidating its underlying disease mechanisms is vital to the development of appropriate therapeutics. We identified differentially expressed genes (DEGs) and differentially spliced genes (DSGs) across the clinical stages of AMD in disease-affected tissue, the macular retina pigment epithelium (RPE)/choroid and the macular neural retina within the same eye. We utilized 27 deeply phenotyped donor eyes (recovered within a 6 h postmortem interval time) from Caucasian donors (60-94 years) using a standardized published protocol. Significant findings were then validated in an independent set of well-characterized donor eyes (n = 85). There was limited overlap between DEGs and DSGs, suggesting distinct mechanisms at play in AMD pathophysiology. A greater number of previously reported AMD loci overlapped with DSGs compared to DEGs between disease states, and no DEG overlap with previously reported loci was found in the macular retina between disease states. Additionally, we explored allele-specific expression (ASE) in coding regions of previously reported AMD risk loci, uncovering a significant imbalance in C3 rs2230199 and CFH rs1061170 in the macular RPE/choroid for normal eyes and intermediate AMD (iAMD), and for CFH rs1061147 in the macular RPE/choroid for normal eyes and iAMD, and separately neovascular AMD (NEO). Only significant DEGs/DSGs from the macular RPE/choroid were found to overlap between disease states. STAT1, validated between the iAMD vs. normal comparison, and AGTPBP1, BBS5, CERKL, FGFBP2, KIFC3, RORα, and ZNF292, validated between the NEO vs. normal comparison, revealed an intricate regulatory network with transcription factors and miRNAs identifying potential upstream and downstream regulators. Findings regarding the complement genes C3 and CFH suggest that coding variants at these loci may influence AMD development via an imbalance of gene expression in a tissue-specific manner. Our study provides crucial insights into the multifaceted genomic underpinnings of AMD (i.e., tissue-specific gene expression changes, potential splice variation, and allelic imbalance), which may open new avenues for AMD diagnostics and therapies specific to iAMD and NEO.

ERp29 Attenuates Nicotine-Induced Endoplasmic Reticulum Stress and Inhibits Choroidal Neovascularization.

Nicotine-induced endoplasmic reticulum (ER) stress in retinal pigment epithelium (RPE) cells is thought to be one pathological mechanism underlying age-related macular degeneration (AMD). ERp29 attenuates tobacco extract-induced ER stress and mitigates tight junction damage in RPE cells. Herein, we aimed to further investigate the role of ERp29 in nicotine-induced ER stress and choroidal neovascularization (CNV). We found that the expression of ERp29 and GRP78 in ARPE-19 cells was increased in response to nicotine exposure. Overexpression of ERp29 decreased the levels of GRP78 and the C/EBP homologous protein (CHOP). Knockdown of ERp29 increased the levels of GRP78 and CHOP while reducing the viability of ARPE-19 cells under nicotine exposure conditions. In the ARPE-19 cell/macrophage coculture system, overexpression of ERp29 decreased the levels of M2 markers and increased the levels of M1 markers. The viability, migration and tube formation of human umbilical vein endothelial cells (HUVECs) were inhibited by conditioned medium from the ERp29-overexpressing group. Moreover, overexpression of ERp29 inhibits the activity and growth of CNV in mice exposed to nicotine in vivo. Taken together, our results revealed that ERp29 attenuated nicotine-induced ER stress, regulated macrophage polarization and inhibited CNV.

Essential Role of XBP1 in Maintaining Photoreceptor Synaptic Integrity in Early Diabetic Retinopathy.

Purpose: Diabetic retinopathy (DR) is a leading cause of blindness in working-age adults characterized by retinal dysfunction and neurovascular degeneration. We previously reported that deletion of X-box binding protein 1 (XBP1) leads to accelerated retinal neurodegeneration in diabetes; however, the mechanisms remain elusive. The goal of this study is to determine the role of XBP1 in the regulation of photoreceptor synaptic integrity in early DR. Methods: Diabetes was induced by streptozotocin in retina-specific XBP1 conditional knockout (cKO) or wild-type (WT) mice to generate diabetic cKO (cKO/DM) or WT/DM mice for comparison with nondiabetic cKO (cKO/NDM) and WT/NDM mice. Retinal morphology, structure, and function were assessed by immunohistochemistry, optical coherence tomography, and electroretinogram (ERG) after 3 months of diabetes. The synapses between photoreceptors and bipolar cells were examined by confocal microscopy, and synaptic integrity was quantified using the QUANTOS algorithm. Results: We found a thinning of the outer nuclear layer and a decline in the b-wave amplitude in dark- and light-adapted ERG in cKO/DM mice compared to all other groups. In line with these changes, cKO mice showed increased loss of synaptic integrity compared to WT mice, regardless of diabetes status. In searching for candidate molecules responsible for the loss of photoreceptor synaptic integrity in diabetic and XBP1-deficient retinas, we found decreased mRNA and protein levels of DLG4/PSD-95 in cKO/DM retina compared to WT/DM. Conclusions: These findings suggest that XBP1 is a crucial regulator in maintaining synaptic integrity and retinal function, possibly through regulation of synaptic scaffold proteins.

Neuronal p58IPK Protects Retinal Ganglion Cells Independently of Macrophage/Microglia Activation in Ocular Hypertension.

p58IPK is a multifaceted endoplasmic reticulum (ER) chaperone and a regulator of eIF2α kinases involved in a wide range of cellular processes including protein synthesis, ER stress response, and macrophage-mediated inflammation. Systemic deletion of p58IPK leads to age-related loss of retinal ganglion cells (RGC) and exacerbates RGC damage induced by ischemia/reperfusion and increased intraocular pressure (IOP), suggesting a protective role of p58IPK in the retina. However, the mechanisms remain elusive. Herein, we investigated the cellular mechanisms underlying the neuroprotection action of p58IPK using conditional knockout (cKO) mouse lines where p58IPK is deleted in retinal neurons (Chx10-p58IPK cKO) or in myeloid cells (Lyz2-p58IPK cKO). In addition, we overexpressed p58IPK by adeno-associated virus (AAV) in the retina to examine the effect of p58IPK on RGC survival after ocular hypertension (OHT) in wild type (WT) mice. Our results show that overexpression of p58IPK by AAV significantly improved RGC survival after OHT in WT mice, suggesting a protective effect of p58IPK on reducing RGC injury. Conditional knockout of p58IPK in retinal neurons or in myeloid cells did not alter retinal structure or cellular composition. However, a significant reduction in the b wave of light-adapted electroretinogram (ERG) was observed in Chx10-p58IPK cKO mice. Deletion of p58IPK in retinal neurons exacerbates RGC loss at 14 days after OHT. In contrast, deficiency of p58IPK in myeloid cells increased the microglia/macrophage activation but had no effect on RGC loss. We conclude that deletion of p58IPK in macrophages increases their activation, but does not influence RGC survival. These results suggest that the neuroprotective action of p58IPK is mediated by its expression in retinal neurons, but not in macrophages. Therefore, targeting p58IPK specifically in retinal neurons is a promising approach for the treatment of neurodegenerative retinal diseases including glaucoma.

Sustained Upregulation of Endothelial Nox4 Mediates Retinal Vascular Pathology in Type 1 Diabetes.

NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in retinal endothelial cells (ECs) and is upregulated under hyperglycemic and hypoxic conditions. However, the role of endothelial Nox4 upregulation in long-term retinal blood vessel damage in diabetic retinopathy (DR) remains undefined. Here, we attempted to address this question using humanized EC-specific Nox4 transgenic (hNox4EC-Tg) and EC-specific Nox4 knockout (Nox4EC-KO) mouse models. Our results show that hNox4EC-Tg mice at age of 10-12 months exhibited increased tortuosity of retinal blood vessels, focal vascular leakage, and acellular capillary formation. In vitro study revealed enhanced apoptosis in brain microvascular ECs derived from hNox4EC-Tg mice, concomitant with increased mitochondrial ROS, elevated lipid peroxidation, decreased mitochondrial membrane potential, and reduced mitochondrial respiratory function. In contrast, EC-specific deletion of Nox4 decreased mitochondrial ROS generation, alleviated mitochondrial damage, reduced EC apoptosis, and protected the retina from acellular capillary formation and vascular hyperpermeability in a streptozotocin-induced diabetes mouse model. These findings suggest that sustained upregulation of Nox4 in the endothelium contributes to retinal vascular pathology in diabetes, at least in part, through impairing mitochondrial function. Normalization of Nox4 expression in ECs may provide a new approach for prevention of vascular injury in DR.

Proteomic Analysis of Retinal Mitochondria-Associated ER Membranes Identified Novel Proteins of Retinal Degeneration in Long-Term Diabetes.

The mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is the physical contact site between the ER and the mitochondria and plays a vital role in the regulation of calcium signaling, bioenergetics, and inflammation. Disturbances in these processes and dysregulation of the ER and mitochondrial homeostasis contribute to the pathogenesis of diabetic retinopathy (DR). However, few studies have examined the impact of diabetes on the retinal MAM and its implication in DR pathogenesis. In the present study, we investigated the proteomic changes in retinal MAM from Long Evans rats with streptozotocin-induced long-term Type 1 diabetes. Furthermore, we performed in-depth bioinformatic analysis to identify key MAM proteins and pathways that are potentially implicated in retinal inflammation, angiogenesis, and neurodegeneration. A total of 2664 unique proteins were quantified using IonStar proteomics-pipeline in rat retinal MAM, among which 179 proteins showed significant changes in diabetes. Functional annotation revealed that the 179 proteins are involved in important biological processes such as cell survival, inflammatory response, and cellular maintenance, as well as multiple disease-relevant signaling pathways, e.g., integrin signaling, leukocyte extravasation, PPAR, PTEN, and RhoGDI signaling. Our study provides comprehensive information on MAM protein changes in diabetic retinas, which is helpful for understanding the mechanisms of metabolic dysfunction and retinal cell injury in DR.

AMD Genomics: Non-Coding RNAs as Biomarkers and Therapeutic Targets.

Age-related macular degeneration (AMD) is a progressive neurodegenerative disease that is the world's leading cause of blindness in the aging population. Although the clinical stages and forms of AMD have been elucidated, more specific prognostic tools are required to determine when patients with early and intermediate AMD will progress into the advanced stages of AMD. Another challenge in the field has been the appropriate development of therapies for intermediate AMD and advanced atrophic AMD. After numerous negative clinical trials, an anti-C5 agent and anti-C3 agent have recently shown promising results in phase 3 clinical trials, in terms of slowing the growth of geographic atrophy, an advanced form of AMD. Interestingly, both drugs appear to be associated with an increased incidence of wet AMD, another advanced form of the disease, and will require frequent intravitreal injections. Certainly, there remains a need for other therapeutic agents with the potential to prevent progression to advanced stages of the disease. Investigation of the role and clinical utility of non-coding RNAs (ncRNAs) is a major advancement in biology that has only been minimally applied to AMD. In the following review, we discuss the clinical relevance of ncRNAs in AMD as both biomarkers and therapeutic targets.

Cellular stress signaling and the unfolded protein response in retinal degeneration: mechanisms and therapeutic implications.

BACKGROUND: The retina, as part of the central nervous system (CNS) with limited capacity for self-reparation and regeneration in mammals, is under cumulative environmental stress due to high-energy demands and rapid protein turnover. These stressors disrupt the cellular protein and metabolic homeostasis, which, if not alleviated, can lead to dysfunction and cell death of retinal neurons. One primary cellular stress response is the highly conserved unfolded protein response (UPR). The UPR acts through three main signaling pathways in an attempt to restore the protein homeostasis in the endoplasmic reticulum (ER) by various means, including but not limited to, reducing protein translation, increasing protein-folding capacity, and promoting misfolded protein degradation. Moreover, recent work has identified a novel function of the UPR in regulation of cellular metabolism and mitochondrial function, disturbance of which contributes to neuronal degeneration and dysfunction. The role of the UPR in retinal neurons during aging and under disease conditions in age-related macular degeneration (AMD), retinitis pigmentosa (RP), glaucoma, and diabetic retinopathy (DR) has been explored over the past two decades. Each of the disease conditions and their corresponding animal models provide distinct challenges and unique opportunities to gain a better understanding of the role of the UPR in the maintenance of retinal health and function. METHOD: We performed an extensive literature search on PubMed and Google Scholar using the following keywords: unfolded protein response, metabolism, ER stress, retinal degeneration, aging, age-related macular degeneration, retinitis pigmentosa, glaucoma, diabetic retinopathy. RESULTS AND CONCLUSION: We summarize recent advances in understanding cellular stress response, in particular the UPR, in retinal diseases, highlighting the potential roles of UPR pathways in regulation of cellular metabolism and mitochondrial function in retinal neurons. Further, we provide perspective on the promise and challenges for targeting the UPR pathways as a new therapeutic approach in age- and disease-related retinal degeneration.

Emerging roles of circular RNAs in retinal diseases.

Retinal disorders are a group of ocular diseases whose onset is associated with a number of aberrant molecular and cellular processes or physical damages that affect retinal structure and function resulting in neural and vascular degeneration in the retina. Current research has primarily focused on delaying retinal disease with minimal success in preventing or reversing neuronal degeneration. In this review, we explore a relatively new field of research involving circular RNAs, whose potential roles as biomarkers and mediators of retinal disease pathogenesis have only just emerged. While knowledge of circular RNAs function is limited given its novelty, current evidence has highlighted their roles as modulators of microRNAs, regulators of gene transcription, and biomarkers of disease development and progression. Here, we summarize how circular RNAs may be implicated in the pathogenesis of common retinal diseases including diabetic retinopathy, glaucoma, proliferative vitreoretinopathy, and age-related macular degeneration. Further, we explore the potential of circular RNAs as novel biomarkers and therapeutic targets for the diagnosis and treatment of retinal diseases.

Activation of ATF4 triggers trabecular meshwork cell dysfunction and apoptosis in POAG.

Primary open angle glaucoma (POAG) is the leading cause of irreversible blindness. Dysfunction of the trabecular meshwork (TM), resulting in decreased outflow of aqueous humor and increased intraocular pressure (IOP), plays an important role in the pathogenesis of POAG. However, the underlying mechanisms still remain unclear. In this study, we demonstrated that the eIF2-α/ATF4/CHOP branch of unfolded protein response (UPR) was activated in human trabecular meshwork cells (HTMCs) upon tert-butyl hydroperoxide (TBHP) exposure. Inhibition of ATF4 ameliorated TBHP-induced apoptosis and inflammatory cytokine production, while ectopic expression of ATF4 increased the expression of endothelial leukocyte adhesion molecule (ELAM)-1 and IL-8 in HTMCs. Furthermore, we found that ATF4 inhibition reduced tunicamycin-induced caspase-3 activation, ROS production, ELAM-1 expression, and HTMCs phagocytosis impairment. By an in vivo study in mice, we showed that overexpression of ATF4 in the TM induced C/EBP homologous protein (CHOP) expression and TM cells apoptosis, contributing to inflammatory cytokine production, and probably IOP elevation. More importantly, upregulation of ATF4 and CHOP, and colocalization of ATF4 with ELAM-1 were found in the TM of POAG patients. These results suggest that ATF4 is a critical mediator of oxidative stress and ER stress-induced TM cell dysfunction and apoptosis in POAG.

Endothelium-specific deletion of Nox4 delays retinal vascular development and mitigates pathological angiogenesis.

NADPH oxidase 4 (Nox4) is a major isoform of NADPH oxidases playing an important role in many biological processes. Previously we have shown that Nox4 is highly expressed in retinal blood vessels and is upregulated in oxygen-induced retinopathy (OIR). However, the exact role of endothelial Nox4 in retinal angiogenesis remains elusive. Herein, using endothelial cell (EC)-specific Nox4 knockout (Nox4EC-KO) mice, we investigated the impact of endothelial Nox4 deletion on retinal vascular development and pathological angiogenesis during OIR. Our results show that deletion of Nox4 in ECs led to retarded retinal vasculature development with fewer, blunted-end tip cells and sparser, dysmorphic filopodia at vascular front, and reduced density of vascular network in superficial, deep, and intermediate layers in postnatal day 7 (P7), P12, and P17 retinas, respectively. In OIR, loss of endothelial Nox4 had no effect on hyperoxia-induced retinal vaso-obliteration at P9 but significantly reduced aberrant retinal neovascularization at P17 and decreased the deep layer capillary density at P25. Ex vivo study confirmed that lack of Nox4 in ECs impaired vascular sprouting. Mechanistically, loss of Nox4 significantly reduced expression of VEGF, p-VEGFR2, integrin αV, angiopoietin-2, and p-ERK1/2, attenuating EC migration and proliferation. Taken together, our results indicate that endothelial Nox4 is important for retinal vascular development and contributes to pathological angiogenesis, likely through regulation of VEGF/VEGFR2 and angiopoietin-2/integrin αV/ERK pathways. In addition, our study suggests that endothelial Nox4 appears to be essential for intraretinal revascularization after hypoxia. These findings call for caution on targeting endothelial Nox4 in ischemic/hypoxic retinal diseases.

Serum pigment epithelium-derived factor: Relationships with cardiovascular events, renal dysfunction, and mortality in the Veterans Affairs Diabetes Trial (VADT) cohort.

BACKGROUND: To determine if serum pigment epithelium-derived factor (PEDF) levels predict cardiovascular events, renal dysfunction and mortality in the Veterans Affairs Diabetes Study (VADT). METHODS: PEDF was evaluated in relation to subsequent cardiovascular outcomes, mortality, and renal dysfunction (defined as urinary albumin creatinine ratio (ACR) ≥300 mg/g), or chronic kidney disease (CKD) stages 3 (eGFR<60 ml/min) or 4 (eGFR<60 and <30 ml/min respectively). PEDF was measured by ELISA on sera from 881 participants collected a median (range) of 1.7 (0-5.0) years post-baseline, and later, from 832 participants 4.0 (1.5-6.9) years post-baseline. RESULTS: In 743 participants, PEDF was measured at both time-points. PEDF increased over time from (mean ±â€¯SD) 10.5 ±â€¯4.03 to 11.0 ±â€¯4.86 ng/ml (paired t-test p = 0.0092). Lower eGFR (p < 0.01), higher serum creatinine (p < 0.01) and urinary ACR (p < 0.01) were associated with increasing PEDF. Multivariate event time models included either one or two follow-up windows (i.e., between first and second PEDF measures; and, when available, from second PEDF measure until study-end). PEDF tertiles were not associated with cardiovascular events, but were significantly associated with all-cause mortality [HR = 2.00 (1.03, 3.89) comparing first to third tertile] in models adjusted for age, minority status, VADT treatment arm and prior cardiovascular event status. Higher PEDF levels also associated with development of kidney dysfunction with adjusted HRs (95% CI comparing third to first PEDF tertiles: 2.74 (1.71, 4.39) for stage 3 CKD; and 3.84 (95% CI: 1.17, 12.5) for stage 4 CKD. CONCLUSIONS: Over 2-years, higher serum PEDF levels predicted advanced nephropathy in patients with type 2 diabetes.

Loss of XBP1 Leads to Early-Onset Retinal Neurodegeneration in a Mouse Model of Type I Diabetes.

Retinal neuronal injury and degeneration is one of the primary manifestations of diabetic retinopathy, a leading cause of vision loss in working age adults. In pathological conditions, including diabetes and some physiological conditions such as aging, protein homeostasis can become disrupted, leading to endoplasmic reticulum (ER) stress. Severe or unmitigated ER stress can lead to cell death, which in retinal neurons results in irreversible loss of visual function. X-box binding protein 1 (XBP1) is a major transcription factor responsible for the adaptive unfolded protein response (UPR) to maintain protein homeostasis in cells undergoing ER stress. The purpose of this study is to determine the role of XBP1-mediated UPR in retinal neuronal survival and function in a mouse model of type 1 diabetes. Using a conditional retina-specific XBP1 knockout mouse line, we demonstrate that depletion of XBP1 in retinal neurons results in early onset retinal function decline, loss of retinal ganglion cells and photoreceptors, disrupted photoreceptor ribbon synapses, and Müller cell activation after induction of diabetes. Our findings suggest an important role of XBP1-mediated adaptive UPR in retinal neuronal survival and function in diabetes.

Loss of X-box binding protein 1 in Müller cells augments retinal inflammation in a mouse model of diabetes.

AIMS/HYPOTHESIS: Müller glia (MG) are major sources of retinal cytokines, and their activation is closely linked to retinal inflammation and vascular leakage in diabetic retinopathy. Previously, we demonstrated that X-box binding protein 1 (XBP1), a transcription factor activated by endoplasmic reticulum (ER) stress in diabetic retinopathy, is involved in regulation of inflammation in retinal endothelial cells. Now, we have explored the role of XBP1 and ER stress in the regulation of MG-derived proinflammatory factors, and their influence on vascular permeability in diabetic retinopathy. METHODS: MG-specific conditional Xbp1 knockout (Xbp1Müller-/-) mice were generated by crossing Xbp1 flox/flox mice with Müller-Cre transgenic mice. Diabetes was modelled by induction with streptozotocin, and retinal vascular permeability was measured with FITC-conjugated dextran 2 months after induction. Primary Müller cells were isolated from Xbp1Müller-/- and Xbp1Müller+/+ mice and exposed to hypoxia and high levels of glucose. Levels of ER-stress and inflammatory factors were examined by real-time PCR, western blotting or immunohistochemistry. RESULTS: Xbp1Müller-/- mice exhibited normal retinal development and retinal function and expressed similar levels of ER-stress and inflammatory genes to Xbp1Müller+/+ littermates. In diabetes-inducing conditions, compared with Xbp1Müller+/+ mice, Xbp1Müller-/- mice had higher mRNA levels of retinal Vegf (also known as Vegfa) and Tnf-α (also known as Tnf) and ER-stress marker genes Grp78 (also known as Hspa5), Atf4, Chop (also known as Ddit3) and Atf6 and higher protein levels of vascular endothelial growth factor (VEGF), TNF-α, phospho-c-Jun N-terminal kinase (JNK), 78 kDa glucose-regulated protein (GRP78), phospho-eukaryotic translation initiation factor (eIF)2α and activating transcription factor (ATF)6. Retinal vascular permeability was significantly higher in diabetic Xbp1Müller-/- mice than in diabetic Xbp1Müller+/+ mice (p < 0.01). Results obtained in vitro with primary Müller cells isolated from Xbp1Müller-/- mice confirmed higher expression levels of inflammatory and ER-stress markers (but not GRP78) than in cells from Xbp1Müller+/+ mice. Moreover, XBP1-deficient Müller cells were more susceptible to high-glucose- or hypoxia-induced ER stress and inflammation than cells from Xbp1Müller+/+ mice. Inhibition of ER stress with chemical chaperones suppressed hypoxia-induced VEGF and TNF-α production in XBP1-deficient Müller cells. CONCLUSIONS/INTERPRETATION: Our results have revealed an important role of XBP1 and ER stress in MG-driven retinal inflammation, and suggest that targeting ER stress may represent a promising approach for the prevention and treatment of diabetic retinopathy.

The unfolded protein response signaling and retinal Müller cell metabolism.

The retina is one of the most energy demanding tissues in the body. Like most neurons in the central nervous system, retinal neurons consume high amounts of adenosine-5'-triphosphate (ATP) to generate visual signal and transmit the information to the brain. Disruptions in retinal metabolism can cause neuronal dysfunction and degeneration resulting in severe visual impairment and even blindness. The homeostasis of retinal metabolism is tightly controlled by multiple signaling pathways, such as the unfolded protein response (UPR), and the close interactions between retinal neurons and other retinal cell types including vascular cells and Müller glia. The UPR is a highly conserved adaptive cellular response and can be triggered by many physiological stressors and pathophysiological conditions. Activation of the UPR leads to changes in glycolytic rate, ATP production, de novo serine synthesis, and the hexosamine biosynthetic pathway, which are considered critical components of Müller glia metabolism and provide metabolic support to surrounding neurons. When these pathways are disrupted, neurodegeneration occurs rapidly. In this review, we summarize recent advance in studies of the UPR in Müller glia and highlight the potential role of the UPR in retinal degeneration through regulation of Müller glia metabolism.

Molecular Chaperone ERp29: A Potential Target for Cellular Protection in Retinal and Neurodegenerative Diseases.

The molecular chaperone endoplasmic reticulum protein 29 (ERp29) plays a critical role in protein folding, trafficking, and secretion. Though ubiquitously expressed, ERp29 is upregulated in response to ER stress and is found at higher levels in certain cell types such as secretory epithelial cells and neurons. As an ER resident protein, ERp29 shares many structural and functional similarities with protein disulfide isomerases, but is not regarded as part of this family due to several key differences. The broad expression and myriad roles of ERp29 coupled with its upregulation via the unfolded protein response (UPR) upon ER stress have implicated ERp29 in a range of cellular processes and diseases. We summarize the diverse activities of ERp29 in protein trafficking, cell survival and apoptosis, and ER homeostasis and highlight a potential role of ERp29 in neuroprotection in retinal and neurodegenerative diseases.

Loss of XBP1 accelerates age-related decline in retinal function and neurodegeneration.

BACKGROUND: Aging is the strongest risk factor for neurodegenerative diseases and extended age results in neuronal degeneration and functional decline in the visual system. Among many contributing factors to age-related deterioration of neurons is an insufficient activation of the Unfolded Protein Response (UPR) in the endoplasmic reticulum (ER) in response to cellular stress. X-box binding protein 1 (XBP1) is a major component of the UPR and is essential for maintaining protein homeostasis and reducing cellular stresses. Herein, we investigate the role of XBP1 in maintaining morphological and functional integrity in retinal neurons during adulthood and the early stages of aging. METHODS: The basal and induced levels of XBP1 activation in the retina were measured in young adult and aged mice. Conditional knockout (cKO) of XBP1 in retinal neurons was achieved by crossing XBP1 floxed mice with a retina specific Cre-recombinase line (Chx10-Cre). Retinal morphology, neuronal populations including photoreceptors, bipolar cells, and retinal ganglion cells (RGCs), synaptic structure, and microglial activation were examined with immunohistochemistry and staining of retinal sections. Retinal function was evaluated with light-adapted (photopic) and dark adapted (scotopic) electroretinograms. Retinal mitochondrial function and metabolism was assessed by Seahorse XFe24 Extracellular Flux Analyzer. RESULTS: The retinas of aged wild type (WT) mice display a significantly reduced basal level of Xbp1s and compromised activation of ER stress response. In XBP1 cKO mice, significant structural degeneration of the retina, evidenced by thinning of retinal layers and a loss of RGCs, and functional defects indicated by diminished photopic and scotopic ERG b-waves are observed at the age of 12-14 months. Furthermore, discontinuous and disorganized synaptic laminae, colocalized with activated microglia, in the inner plexiform layer is found in the XBP1 cKO retinas. In addition, cKO mice demonstrate a significant increase in ectopic synapses between bipolar cells and photoreceptors, which is strikingly similar to WT mice at 20-24 months of age. These changes are associated with defective retinal glycolysis while mitochondrial respiratory function appears normal in the cKO retina. CONCLUSIONS: XBP1 cKO mice at 12-14 months of age show significant structural, functional, and metabolic deficits that closely resemble WT mice twice that age. Our findings suggest that the absence of XBP1, a critical component of the UPR, accelerates age-related retinal neurodegeneration.