Potential Combination Drug Therapy to Prevent Redox Stress and Mitophagy Dysregulation in Retinal Müller Cells under High Glucose Conditions: Implications for Diabetic Retinopathy.

Chronic hyperglycemia-induced thioredoxin-interacting protein (TXNIP) expression, associated oxidative/nitrosative stress (ROS/RNS), and mitochondrial dysfunction play critical roles in the etiology of diabetic retinopathy (DR). However, there is no effective drug treatment to prevent or slow down the progression of DR. The purpose of this study is to examine if a combination drug treatment targeting TXNIP and the mitochondria-lysosome pathway prevents high glucose-induced mitochondrial stress and mitophagic flux in retinal Müller glial cells in culture, relevant to DR. We show that diabetes induces TXNIP expression, redox stress, and Müller glia activation (gliosis) in rat retinas when compared to non-diabetic rat retinas. Furthermore, high glucose (HG, 25 mM versus low glucose, LG 5.5 mM) also induces TXNIP expression and mitochondrial stress in a rat retinal Müller cell line, rMC1, in in vitro cultures. Additionally, we develop a mitochondria-targeted mCherry and EGFP probe tagged with two tandem COX8a mitochondrial target sequences (adenovirus-CMV-2×mt8a-CG) to examine mitophagic flux in rMC1. A triple drug combination treatment was applied using TXNIP-IN1 (which inhibits TXNIP interaction with thioredoxin), Mito-Tempo (mitochondrial anti-oxidant), and ML-SA1 (lysosome targeted activator of transient calcium channel MCOLN1/TRPML1 and of transcription factor TFEB) to study the mitochondrial-lysosomal axis dysregulation. We found that HG induces TXNIP expression, redox stress, and mitophagic flux in rMC1 versus LG. Treatment with the triple drug combination prevents mitophagic flux and restores transcription factor TFEB and PGC1α nuclear localization under HG, which is critical for lysosome biosynthesis and mitogenesis, respectively. Our results demonstrate that 2×mt8a-CG is a suitable probe for monitoring mitophagic flux, both in live and fixed cells in in vitro experiments, which may also be applicable to in vivo animal studies, and that the triple drug combination treatment has the potential for preventing retinal injury and disease progression in diabetes.

Mitophagy, Ferritinophagy and Ferroptosis in Retinal Pigment Epithelial Cells Under High Glucose Conditions: Implications for Diabetic Retinopathy and Age-Related Retinal Diseases.

Diabetic retinopathy (DR) is a devastating disease leading to blindness among majority of working adults around the globe. Nonetheless, an effective treatment or cure for the disease is still to be achieved. This is because the cellular and molecular mechanisms of DR are complex and not fully understood yet. In this article, we describe how high glucose induced TXNIP upregulation and associated redox stress may cause mitochondrial dysfunction, mitophagy, ferritinophagy (iron release by autophagy) and lysosome destabilization. Labile irons react with hydrogen peroxide (H2O2) to generate hydroxyl radicals (.OH) by the Fenton reaction and cause membrane phospholipid peroxidation due to reduction in glutathione (GSH) level and glutathione peroxidase 4 (GPX4) activity, which cause ferroptosis, a recently identified non-apoptotic cell death mechanism. We used in this study a retinal pigment epithelial cell line, ARPE- 19 and exposed it to high glucose in in vitro cultures to highlight some of the intricacies of these cellular processes, which may be relevant to the pathogenesis of DR and age-related retinal neurodegenerative diseases, such as age-related macular degeneration, AMD.

TXNIP mediates high glucose-induced mitophagic flux and lysosome enlargement in human retinal pigment epithelial cells.

Thioredoxin-interacting protein (TXNIP) plays a critical role in oxidative stress, inflammation, apoptosis and the pathogenesis of diabetic retinopathy (DR). However, the role of TXNIP in high glucose-induced retinal pigment epithelium (RPE) dysfunction is still unknown. Here, we show that high glucose (HG; 25 mM,) significantly increases TXNIP expression at both the mRNA and protein levels when compared to low glucose (LG; 5.5 mM) in a human RPE cell line (ARPE-19) and primary human RPE (HRPE) cells. TXNIP upregulation is associated with mitochondrial membrane depolarization, fragmentation and mitophagic flux to lysosomes. We used confocal live-cell imaging of RPE cells expressing mt-Keima, a coral protein that emits green light in mitochondria (alkaline or neutral pH) and red light in the acidic lysosome, to measure mitophagic flux. We observed an elongated mitochondrial network of green mt-Keima under LG, which is fragmented in HG. Red mt-Keima accumulates in lysosomes as small punctate aggregations under LG in both ARPE-19 and HRPE cells, whereas they are significantly enlarged (two- to threefold) under HG. Lysosomal enlargement under HG is further illustrated by lysosomal membrane protein LAMP1-mCherry expression in both ARPE-19 and HRPE cells. Furthermore, HG causes lysosomal cathepsin L inactivation and pro-inflammatory caspase-1 activation in ARPE-19 cells. TXNIP knockdown by shRNA prevents mitochondrial fragmentation, mitophagic flux and lysosome enlargement under HG. In addition, antioxidant N-acetylcysteine (NAC) and Amlexanox (Amlx), an inhibitor of protein kinase TBK1 and of the mitophagic adaptors Optineurin (Optn) and Sequestosome 1 (p62/SQSTM1), prevent mitophagic flux and lysosome enlargement. These results suggest that TXNIP mediates several deleterious effects of high glucose on RPE, which may be implicated in the development of DR.

The Role of Txnip in Mitophagy Dysregulation and Inflammasome Activation in Diabetic Retinopathy: A New Perspective.

Mitochondria are responsible for bioenergetics, metabolism and apoptosis signals in health and disease. The retina being a part of the central nervous system consumes large amounts of glucose and oxygen to generate ATP via the mitochondrial oxidative phosphorylation for its phototransduction and visual function. During ATP generation, electrons leak from the mitochondrial electron transport chain, which is captured by molecular oxygen to produce reactive oxygen species (ROS). These mtROS damage mitochondrial proteins, mtDNA, and membrane lipids and release them in the cytosol. Mitochondrial components are recognized as danger-associated molecular patterns (DAMPS) by cytosolic pattern recognition receptors such as NOD-like receptors, NLRP3 inflammasomes. They process pro-caspase-1 to active caspase-1, which cleaves pro-inflammatory IL-1ß o mature IL-1ß causing inflammation and cell death by pyroptosis. To counter the damaging action of mtROS and inflammasomes in fully differentiated cells in the retina, the removal of the damaged and dysfunctional mitochondria by a double-membrane autophagic process via lysosomal degradation called mitophagy is critical for mitochondrial homeostasis and cell survival. Nonetheless, under chronic diseases including diabetic retinopathy (DR), mitophagy dysregulation and NLRP3 inflammasome activation exist, which cause premature cell death and disease progression. Recently, the thioredoxin-interacting protein TXNIP, which is strongly induced by diabetes and inhibits anti-oxidant function of thioredoxin, has been implicated in mitochondrial dysfunction, mitophagic dysregulation and NLRP3 inflammasome activation in DR. Therefore, TXNIP silencing or pharmacological inhibition may normalize mitophagic flux and NLRP3 inflammasome activation, which will prevent or slow down the progression of DR.

Critical role of TXNIP in oxidative stress, DNA damage and retinal pericyte apoptosis under high glucose: implications for diabetic retinopathy.

Diabetic retinopathy (DR) is characterized by early loss of retinal capillary pericytes and microvascular dysfunction. We recently showed that pro-oxidative stress and pro-apoptotic thioredoxin interacting protein (TXNIP) is significantly up-regulated in rat retinas in experimental diabetes and mediates inflammation and apoptosis. Therefore, we hypothesize here that TXNIP up-regulation in pericyte plays a causative role in oxidative stress and apoptosis under sustained high glucose exposure in culture. We maintained a rat retinal capillary pericyte cell line (TR-rPCT1) for 5 days under low glucose (LG, 5.5mM) or high glucose (HG, 25 mM) with or without anti-oxidant N-acetylcysteine (5mM, NAC), Azaseine (2 µM, AzaS), an inhibitor of TXNIP, and TXNIP siRNA (siTXNIP3, 20 nM). The results show that HG increases TXNIP expression in TR-rPCT1, which correlates positively with ROS generation, protein S-nitrosylation, and pro-apoptotic caspase-3 activation. Furthermore, pericyte apoptosis is demonstrated by DNA fragmentation (alkaline comet assay) and a reduction in MTT survival assay. Treatment of TR-rPCT1 with NAC or an inhibition of TXNIP by AzaS or siTXNIP3 each reduces HG-induced ROS, caspase-3 activation and DNA damage demonstrating that TXNIP up-regulation under chronic hyperglycemia is critically involved in cellular oxidative stress, DNA damage and retinal pericyte apoptosis. Thus, TXNIP represents a novel gene and drug target to prevent pericyte loss and progression of DR.

TXNIP links innate host defense mechanisms to oxidative stress and inflammation in retinal Muller glia under chronic hyperglycemia: implications for diabetic retinopathy.

Thioredoxin Interacting Protein (TXNIP) mediates retinal inflammation, gliosis, and apoptosis in experimental diabetes. Here, we investigate the temporal response of Muller glia to high glucose (HG) and TXNIP expression using a rat Muller cell line (rMC1) in culture. We examined if HG-induced TXNIP expression evokes host defense mechanisms in rMC1 in response to metabolic abnormalities. HG causes sustained up-regulation of TXNIP (2 h to 5 days), ROS generation, ATP depletion, ER stress, and inflammation. Various cellular defense mechanisms are activated by HG: (i) NLRP3 inflammasome, (ii) ER stress response (sXBP1), (iii) hypoxic-like HIF-1α induction, (iv) autophagy/mitophagy, and (v) apoptosis. We also found in vivo that streptozocin-induced diabetic rats have higher retinal TXNIP and innate immune response gene expression than normal rats. Knock down of TXNIP by intravitreal siRNA reduces inflammation (IL-1ß) and gliosis (GFAP) in the diabetic retina. TXNIP ablation in vitro prevents ROS generation, restores ATP level and autophagic LC3B induction in rMC1. Thus, our results show that HG sustains TXNIP up-regulation in Muller glia and evokes a program of cellular defense/survival mechanisms that ultimately lead to oxidative stress, ER stress/inflammation, autophagy and apoptosis. TXNIP is a potential target to ameliorate blinding ocular complications of diabetic retinopathy.

GSK-3ß/CREB axis mediates IGF-1-induced ECM/adhesion molecule expression, cell cycle progression and monolayer permeability in retinal capillary endothelial cells: Implications for diabetic retinopathy.

Various growth factors and cytokines are implicated in endothelial dysfunction and blood-retinal barrier (BRB) breakdown in early diabetic retinopathy (DR). However, cellular and molecular mechanisms that may underlie the pathology of DR are not fully understood yet. We therefore examined the effect of insulin-like growth factor (IGF)-1 on ECM/adhesion molecule expression, cell cycle regulation and monolayer permeability in an endothelial cell line (TR-iBRB2). We investigate whether the action of IGF-1 (1) involves glycogen synthase kinase 3beta (GSK-3ß) and cAMP responsive transcription factor (CREB) and (2) alters ECM/adhesion molecule gene expression. Treatment of TR-iBRB2 cell with IGF-1 (100ng/ml for 0-24h) increases phosphorylation of (i) Akt Thr308, and its substrates including GSK-3ß at Ser9, which inactivates its kinase function, and (ii) CREB at Ser133 (activation). These phosphorylations correlate positively with enhanced expression of CREB targets such as ECM protein fibronectin and cell cycle progression factor cyclin D1. However, stable transfection of a mutant GSK3ß(S9A) or a dominant negative K-CREB in TR-iBRB2 prevents IGF-1-induced fibronectin and cyclin D1 expression. Furthermore, IGF-1 reduces the level of intercellular adherence molecule VE-cadherin and increases monolayer permeability in TR-iBRB2 cells when measured by FITC-dextran leakage. The effect of IGF-1 on VE-cadherin and membrane permeability is absent in TR-iBRB2 cells expressing the GSK-3ß(S9A). Similarly, K-CREB reverses IGF-1 down-regulation of VE-cadherin and up-regulation of fibronectin. These results indicate that GSK-3ß/CREB axis alters ECM/adhesion molecule expression and cell cycle progression in retinal endothelial cells, and may potentially contribute to endothelial dysfunction and BRB leakage in DR.

RAGE-TXNIP axis is required for S100B-promoted Schwann cell migration, fibronectin expression and cytokine secretion.

During peripheral nerve injury, Schwann cells (SCs) adopt a migratory phenotype and remodel the extracellular matrix and provide a supportive activity for neuron regeneration. SCs synthesize neurotrophic factors and cytokines that are crucial for the repair of the injured nerve. The receptor for advanced glycation end products (RAGE) and its ligand S100B, which are secreted by SCs, are required for the repair of the injured peripheral nerve in vivo. However, the precise intracellular pathways involved have not been completely elucidated. Here, we show that RAGE-induced S100B secretion involves the recruitment of S100B in lipid rafts and caveolae. Moreover, we demonstrate for the first time that RAGE induces the expression of thioredoxin interacting protein (TXNIP) in SCs and the injured sciatic nerve in vivo. TXNIP is involved in the activation of p38 MAPK, CREB and NFκB in SCs. TXNIP silencing partially inhibits RAGE-induced SC migration and completely abolishes RAGE-induced fibronectin and IL-1ß expression. Our results support a model in which TXNIP mediates in part RAGE-induced SC migration and is required for the expression of provisional ECM and pro-inflammatory IL-1ß. We provide new insight on the role of the SC RAGE-TXNIP axis in the repair of injured peripheral nerves.

Thioredoxin interacting protein (TXNIP) induces inflammation through chromatin modification in retinal capillary endothelial cells under diabetic conditions.

Chronic hyperglycemia and activation of receptor for advanced glycation end products (RAGE) are known risk factors for microvascular disease development in diabetic retinopathy. Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of antioxidant thioredoxin (TRX), plays a causative role in diabetes and its vascular complications. Herein we investigate whether HG and RAGE induce inflammation in rat retinal endothelial cells (EC) under diabetic conditions in culture through TXNIP activation and whether epigenetic mechanisms play a role in inflammatory gene expression. We show that RAGE activation by its ligand S100B or HG treatment of retinal EC induces the expression of TXNIP and inflammatory genes such as Cox2, VEGF-A, and ICAM1. TXNIP silencing by siRNA impedes RAGE and HG effects while stable over-expression of a cDNA for human TXNIP in EC elevates inflammation. p38 MAPK-NF-kappaB signaling pathway and histone H3 lysine (K) nine modifications are involved in TXNIP-induced inflammation. Chromatin immunoprecipitation (ChIP) assays reveal that TXNIP over-expression in EC abolishes H3K9 tri-methylation, a marker for gene inactivation, and increases H3K9 acetylation, an indicator of gene induction, at proximal Cox2 promoter bearing the NF-kappaB-binding site. These findings have important implications toward understanding the molecular mechanisms of ocular inflammation and endothelial dysfunction in diabetic retinopathy.