Neutrophils in Ocular Diseases.

Neutrophils, traditionally viewed as first responders to infection or tissue damage, exhibit dynamic and diverse roles in ocular health and disease. This review elaborates on previous findings that showed how neutrophils contribute to ocular diseases. In ocular infections, neutrophils play a pivotal role in host defense by orchestrating inflammatory responses to combat pathogens. Furthermore, in optic nerve neuropathies and retinal degenerative diseases like age-related macular degeneration (AMD) and diabetic retinopathy (DR), neutrophils are implicated in neuroinflammation and tissue damage owing to their ability to undergo neutrophil extracellular trap formation (NETosis) and secretion of inflammatory molecules. Targeting neutrophil-dependent processes holds promise as a therapeutic strategy, offering potential avenues for intervention in ocular infections, cancers, and retinal degenerative diseases. Understanding the multifaceted roles of neutrophils in ocular diseases is crucial for developing targeted therapies to improve patient outcomes.

The role of lipocalin-2 in age-related macular degeneration (AMD).

Lipocalins are a family of secreted adipokines which play important roles in various biological processes. Lipocalin-2 (LCN-2) has been shown to be involved in acute and chronic inflammation. This particular protein is critical in the pathogenesis of several diseases including cancer, diabetes, obesity, and multiple sclerosis. Herein, we discuss the general molecular basis for the involvement of LCN-2 in acute infections and chronic disease progression and also ascertain the probable role of LCN-2 in ocular diseases, particularly in age-related macular degeneration (AMD). We elaborate on the signaling cascades which trigger LCN-2 upregulation in AMD and suggest therapeutic strategies for targeting such pathways.

A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for In Vitro Studies in AMD.

Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ. These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.

Melatonin as the Possible Link Between Age-Related Retinal Regeneration and the Disrupted Circadian Rhythm in Elderly.

The association between age-related macular degeneration (AMD) and biological rhythms has been insufficiently studied; however there are several reasons to believe that impairment in circadian rhythm may affect incidence and pathogenesis of AMD. The current understanding of AMD pathology is based on age-related, cumulative oxidative damage to the retinal pigmented epithelium (RPE) partially due to impaired clearance of phagocytosed photoreceptor outer segments. In higher vertebrates, phagocytosis of the outer segments is synchronized by circadian rhythms and occurs shortly after dawn, followed by lysosomal-mediated clearance. Aging has been shown to be associated with the changes in circadian rhythmicity of melatonin production, which can be a major factor contributing to the impaired balance between phagocytosis and clearance and increased levels of reactive oxygen species resulting in degenerative changes in the retina. This minireview summarizes studies linking AMD with melatonin production and discusses challenges and perspectives of this area of research.

Activating the AKT2-nuclear factor-κB-lipocalin-2 axis elicits an inflammatory response in age-related macular degeneration.

Age-related macular degeneration (AMD) is a complex and progressive degenerative eye disease resulting in severe loss of central vision. Recent evidence indicates that immune system dysregulation could contribute to the development of AMD. We hypothesize that defective lysosome-mediated clearance causes accumulation of waste products in the retinal pigmented epithelium (RPE), activating the immune system and leading to retinal tissue injury and AMD. We have generated unique genetically engineered mice in which lysosome-mediated clearance (both by phagocytosis and autophagy) in RPE cells is compromised, causing the development of features of early AMD. Our recent data indicate a link between lipocalin-2 (LCN-2) and the inflammatory responses induced in this mouse model. We show that nuclear factor-κB (NF-κB) and STAT-1 may function as a complex in our animal model system, together controlling the upregulation of LCN-2 expression in the retina and stimulating an inflammatory response. This study revealed increased infiltration of LCN-2-positive neutrophils in the choroid and retina of early AMD patients as compared with age-matched controls. Our results demonstrate that, both in our animal model and in human AMD, the AKT2-NF-κB-LCN-2 signalling axis is involved in activating the inflammatory response, making this pathway a potential target for AMD treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ßA3/A1-crystallin and persistent fetal vasculature (PFV) disease of the eye.

BACKGROUND: Persistent fetal vasculature (PFV) is a human disease in which the fetal vasculature of the eye fails to regress normally. The fetal, or hyaloid, vasculature nourishes the lens and retina during ocular development, subsequently regressing after formation of the retinal vessels. PFV causes serious congenital pathologies and is responsible for as much as 5% of blindness in the United States. SCOPE OF REVIEW: The causes of PFV are poorly understood, however there are a number of animal models in which aspects of the disease are present. One such model results from mutation or elimination of the gene (Cryba1) encoding ßA3/A1-crystallin. In this review we focus on the possible mechanisms whereby loss of functional ßA3/A1-crystallin might lead to PFV. MAJOR CONCLUSIONS: Cryba1 is abundantly expressed in the lens, but is also expressed in certain other ocular cells, including astrocytes. In animal models lacking ßA3/A1-crystallin, astrocyte numbers are increased and they migrate abnormally from the retina to ensheath the persistent hyaloid artery. Evidence is presented that the absence of functional ßA3/A1-crystallin causes failure of the normal acidification of endolysosomal compartments in the astrocytes, leading to impairment of certain critical signaling pathways, including mTOR and Notch/STAT3. GENERAL SIGNIFICANCE: The findings suggest that impaired endolysosomal signaling in ocular astrocytes can cause PFV disease, by adversely affecting the vascular remodeling processes essential to ocular development, including regression of the fetal vasculature. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Lysosomes: Regulators of autophagy in the retinal pigmented epithelium.

The retinal pigmented epithelium (RPE) is critically important to retinal homeostasis, in part due to its very active processes of phagocytosis and autophagy. Both of these processes depend upon the normal functioning of lysosomes, organelles which must fuse with (auto)phagosomes to deliver the hydrolases that effect degradation of cargo. It has become clear that signaling through mTOR complex 1 (mTORC1), is very important in the regulation of lysosomal function. This signaling pathway is becoming a target for therapeutic intervention in diseases, including age-related macular degeneration (AMD), where lysosomal function is defective. In addition, our laboratory has been studying animal models in which the gene (Cryba1) for ßA3/A1-crystallin is deficient. These animals exhibit impaired lysosomal clearance in the RPE and pathological signs that are similar to some of those seen in AMD patients. The data demonstrate that ßA3/A1-crystallin localizes to lysosomes in the RPE and that it is a binding partner of V-ATPase, the proton pump that acidifies the lysosomal lumen. This suggests that ßA3/A1-crystallin may also be a potential target for therapeutic intervention in AMD. In this review, we focus on effector molecules that impact the lysosomal-autophagic pathway in RPE cells.

Modulation of V-ATPase by ßA3/A1-Crystallin in Retinal Pigment Epithelial Cells.

We have previously demonstrated that ßA3/A1-crystallin, a member of the ß/γ-crystallin superfamily, is expressed in the astrocytes and retinal pigment epithelial (RPE) cells of the eye. In order to understand the physiological functions of ßA3/A1-crystallin in RPE cells, we generated conditional knockout (cKO) mice where Cryba1, the gene encoding ßA3/A1-crystallin, is deleted specifically from the RPE using the Cre-loxP system. By utilizing the cKO model, we have shown that this protein is required by RPE cells for proper lysosomal degradation of photoreceptor outer segments (OS) that have been internalized in phagosomes and also for the proper functioning of the autophagy process. We also reported that ßA3/A1-crystallin is trafficked to lysosomes, where it regulates endolysosomal acidification by modulating the activity of the lysosomal V-ATPase complex. Our results show that the V-ATPase activity in cKO RPE is significantly lower than WT RPE. Since, V-ATPase is important for regulating lysosomal pH, we noticed that endolysosomal pH was higher in the cKO cells compared to the WT cells. Increased lysosomal pH in cKO RPE is also associated with reduced Cathepsin D activity. Cathepsin D is a major lysosomal aspartic protease involved in the degradation of the OS and hence we believe that reduced proteolytic activity contributes to impaired degradation of OS in the cKO RPE. Reduced lysosomal activity in the cKO RPE also contributes to the incomplete degradation of the autophagosomes. Our results also suggest that ßA3/A1-crystallin regulates V-ATPase activity by binding to the V0 subunit of the V-ATPase complex. Taken together, these results suggest a novel mechanism by which ßA3/A1-crystallin regulates lysosomal function by modulating the activity of V-ATPase.

Expression pattern of an evolutionarily conserved splice variant in the rat Tacc2 gene.

The transforming acidic coiled-coil containing protein 2 (Tacc2) gene and its paralogs, Tacc1 and Tacc3 encode proteins that are associated with the centrosome and involved in microtubule assembly during the cell cycle. Tacc2 produces several splice variants, which are poorly characterized, especially in the rat. Characterization of the temporal/spatial expression patterns of these isoforms would be useful in understanding their distinct and overlapping functions. By comparative sequence analyses of Tacc2 in multiple species, we identified a third splice variant in rat, which is much shorter in size (1,021 aa) than the longest isoform (2,834 aa). This newly identified Tacc2 splice variant (isoform 3) uses a distinct first exon and generates a different open reading frame. Although Isoform 3 is expressed predominantly during developmental stages, the long Tacc2 isoform (isoform 1) is distributed mainly in adult tissues. Multiple protein sequence analyses revealed that Tacc2 Isoform 3 could be the ancient form, as it is conserved in mammals, birds, and amphibians; whereas the long Tacc2 isoforms may have evolved in the mammalian lineage by adding exons toward the 5' region of the ancient isoform.

AKT2-mediated lysosomal dysfunction promotes secretory autophagy in retinal pigment epithelium (RPE) cells.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with the non-neovascular or atrophic form being the most common. Current treatment options are limited, emphasizing the urgent need for new therapeutic strategies. Our key finding is that increased levels of AKT2 in the RPE cells impair lysosomal function and trigger secretory autophagy; a non-canonical macroautophagy/autophagy pathway where cellular materials are released via the plasma membrane rather than being degraded by lysosomes. We showed that this process involves a protein complex, AKT2-SYTL1-TRIM16-SNAP23, releasing factors contributing to drusen biogenesis, a clinical hallmark of AMD development. Importantly, SIRT5 can inhibit this pathway, potentially offering a protective effect. Understanding mechanisms by which this non-canonical autophagy pathway promotes extracellular waste accumulation could provide new insights into drusen biogenesis. Future therapies for atrophic AMD could focus on regulating secretory autophagy or manipulating proteins involved in this process.

ßA3/A1-crystallin is an epigenetic regulator of histone deacetylase 3 (HDAC3) in the retinal pigmented epithelial (RPE) cells.

The retinal pigmented epithelial (RPE) cells maintain retinal homeostasis, and alterations in their function contribute to non-exudative age-related macular degeneration (AMD) 1,2 . Here, we explore the intricate relationship between RPE cells, epigenetic modifications, and the development of AMD. Importantly, the study reveals a substantial decrease in histone deacetylase 3 (HDAC3) activity and elevated histone acetylation in the RPE of human AMD donor eyes. To investigate epigenetic mechanisms in AMD development, we used a mouse model with RPE-specific Cryba1 knockout 3-5 , revealing that the loss of ßA3/A1-crystallin selectively reduces HDAC3 activity, resulting in increased histone acetylation. ßA3/A1-crystallin activates HDAC3 by facilitating its interaction with the casein kinase II (CK2) and phosphorylating HDAC3, as well as by regulating intracellular InsP6 (phytic acid) levels, required for activating HDAC3. These findings highlight a novel function of ßA3/A1-crystallin as an epigenetic regulator of HDAC3 in the RPE cells and provide insights into potential therapeutic strategies in non-exudative AMD.

The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in non-neovascular AMD.

Non-neovascular or dry age-related macular degeneration (AMD) is a multi-factorial disease with degeneration of the aging retinal-pigmented epithelium (RPE). Lysosomes play a crucial role in RPE health via phagocytosis and autophagy, which are regulated by transcription factor EB/E3 (TFEB/E3). Here, we find that increased AKT2 inhibits PGC-1α to downregulate SIRT5, which we identify as an AKT2 binding partner. Crosstalk between SIRT5 and AKT2 facilitates TFEB-dependent lysosomal function in the RPE. AKT2/SIRT5/TFEB pathway inhibition in the RPE induced lysosome/autophagy signaling abnormalities, disrupted mitochondrial function and induced release of debris contributing to drusen. Accordingly, AKT2 overexpression in the RPE caused a dry AMD-like phenotype in aging Akt2 KI mice, as evident from decline in retinal function. Importantly, we show that induced pluripotent stem cell-derived RPE encoding the major risk variant associated with AMD (complement factor H; CFH Y402H) express increased AKT2, impairing TFEB/TFE3-dependent lysosomal function. Collectively, these findings suggest that targeting the AKT2/SIRT5/TFEB pathway may be an effective therapy to delay the progression of dry AMD.

Mutation in the ßA3/A1-crystallin gene impairs phagosome degradation in the retinal pigmented epithelium of the rat.

Phagocytosis of the shed outer segment discs of photoreceptors is a major function of the retinal pigmented epithelium (RPE). We demonstrate for the first time that ßA3/A1-crystallin, a major structural protein of the ocular lens, is expressed in RPE cells. Further, by utilizing the Nuc1 rat, in which the ßA3/A1-crystallin gene is mutated, we show that this protein is required by RPE cells for proper degradation of outer segment discs that have been internalized in phagosomes. We also demonstrate that in wild-type RPE, ßA3/A1-crystallin is localized to the lysosomes. However, in the Nuc1 RPE, ßA3/A1-crystallin fails to translocate to the lysosomes, perhaps because misfolding of the mutant protein masks sorting signals required for proper trafficking. The digestion of phagocytized outer segments requires a high level of lysosomal enzyme activity, and cathepsin D, the major enzyme responsible for proteolysis of the outer segments, is decreased in mutant RPE cells. Interestingly, our results also indicate a defect in the autophagy process in the Nuc1 RPE, which is probably also linked to impaired lysosomal function, because phagocytosis and autophagy might share common mechanisms in degradation of their targets. ßA3/A1-crystallin is a novel lysosomal protein in RPE, essential for degradation of phagocytosed material.

Thermal Shift Assay in Ferroptosis.

Ferroptosis is a recently described process of cell death that is dependent on unregulated cellular iron accumulation with induction of oxidative stress. Ferroptosis has been linked to several human diseases; therefore, investigations aimed at better understanding the pathway and elucidating avenues for future drug development are warranted. Current assays that target ferroptosis/oxidative stress in cells is limited to western blotting and imaging techniques, and unfortunately provide only a broad understanding that is insufficient to effectively assess novel drugs (ligands). Specifically, these assays do not provide insights about ligand interactions with specific proteins associated with these processes. Herein, we discuss a cell-based thermal shift assay that enables screening of ligands under specific cellular conditions for targeting ferroptosis and/or oxidative stress pathways. These data would provide detailed preliminary evidence required for drug development that targets this pathway.

Mitophagy in Astrocytes Is Required for the Health of Optic Nerve.

Mitochondrial dysfunction in astrocytes has been implicated in the development of various neurological disorders. Mitophagy, mitochondrial autophagy, is required for proper mitochondrial function by preventing the accumulation of damaged mitochondria. The importance of mitophagy, specifically in the astrocytes of the optic nerve (ON), has been little studied. We introduce an animal model in which two separate mutations act synergistically to produce severe ON degeneration. The first mutation is in Cryba1, which encodes ßA3/A1-crystallin, a lens protein also expressed in astrocytes, where it regulates lysosomal pH. The second mutation is in Bckdk, which encodes branched-chain ketoacid dehydrogenase kinase, which is ubiquitously expressed in the mitochondrial matrix and involved in the catabolism of the branched-chain amino acids. BCKDK is essential for mitochondrial function and the amelioration of oxidative stress. Neither of the mutations in isolation has a significant effect on the ON, but animals homozygous for both mutations (DM) exhibit very serious ON degeneration. ON astrocytes from these double-mutant (DM) animals have lysosomal defects, including impaired mitophagy, and dysfunctional mitochondria. Urolithin A can rescue the mitophagy impairment in DM astrocytes and reduce ON degeneration. These data demonstrate that efficient mitophagy in astrocytes is required for ON health and functional integrity.

Aberrant Akt2 signaling in the RPE may contribute to retinal fibrosis process in diabetic retinopathy.

Diabetic Retinopathy (DR) is a complication of diabetes that causes blindness in adults. Retinal fibrosis is closely associated with developing proliferative diabetic retinopathy (PDR). Clinical studies have shown that fibrotic membranes exhibit uncontrolled growth in PDR and contribute to retinal detachment from RPE cells, ultimately leading to vision loss. While anti-VEGF agents and invasive laser treatments are the primary treatments for PDR, retinal fibrosis has received minimal attention as a potential target for therapeutic intervention. Therefore, to investigate the potential role of Akt2 in the diabetes-induced retinal fibrosis process, we generated RPE-specific Akt2 conditional knockout (cKO) mice and induced diabetes in these mice and Akt2fl/fl control mice by intraperitoneal injection of streptozotocin. After an 8-month duration of diabetes (10 months of age), the mice were euthanized and expression of tight junction proteins, epithelial-mesenchymal transition (EMT), and fibrosis markers were examined in the RPE. Diabetes induction in the floxed control mice decreased levels of the RPE tight junction protein ZO-1 and adherens junction proteins occludin and E-cadherin; these decreases were rescued in Akt2 cKO diabetic mice. Loss of Akt2 also inhibited diabetes-induced elevation of RNA and protein levels of the EMT markers Snail/Slug and Twist1 in the RPE as compared to Akt2fl/fl diabetic mice. We also found that in Akt2 cKO mice diabetes-induced increase of fibrosis markers, including collagen IV, Connective tissue growth factor (CTGF), fibronectin, and alpha-SMA was attenuated. Furthermore, we observed that high glucose-induced alterations in EMT and fibrosis markers in wild-type (WT) RPE explants were rescued in the presence of PI3K and ERK inhibitors, indicating diabetes-induced retinal fibrosis may be mediated via the PI3K/Akt2/ERK signaling, which could provide a novel target for DR therapy.

Sustained systemic inflammation increases autophagy and induces EMT/fibrotic changes in mouse liver cells: Protection by melatonin.

The unending lifestyle stressors along with genetic predisposition, environmental factors and infections have pushed the immune system into a state of constant activity, leading to unresolved inflammation and increased vulnerability to chronic diseases. Liver fibrosis, an early-stage liver condition that increases the risk of developing liver diseases like cirrhosis and hepatocellular carcinoma, is among the various diseases linked to inflammation that dominate worldwide morbidity and mortality. We developed a mouse model with low-grade lipopolysaccharide (LPS) exposure that shows hepatic damage and a pro-inflammatory condition in the liver. We show that inflammation and oxidative changes increase autophagy in liver cells, a degradation process critical in maintaining cellular homeostasis. Our findings from in vivo and in vitro studies also show that induction of both inflammation and autophagy trigger epithelial-mesenchymal transition (EMT) and pro-fibrotic changes in hepatocytes. Inhibiting the inflammatory pathways with a naturally occurring NF-κB inhibitor and antioxidant, melatonin, could assuage the changes in autophagy and activation of EMT/fibrotic pathways in hepatocytes. Taken together, this study shows a pathway linking inflammation and autophagy which could be targeted for future drug development to delay the progression of liver fibrosis.

Increased LCN2 (lipocalin 2) in the RPE decreases autophagy and activates inflammasome-ferroptosis processes in a mouse model of dry AMD.

In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis. LCN2 binds to ATG4B to form an LCN2-ATG4B-LC3-II complex, thereby regulating ATG4B activity and LC3-II lipidation. Thus, increased LCN2 reduced autophagy flux. Moreover, RPE cells from cryba1 KO, as well as sting1 KO and Sting1Gt mutant mice (models with abnormal iron chelation), showed decreased autophagy flux and increased LCN2, indicative of CGAS- and STING1-mediated inflammasome activation. Live cell imaging of RPE cells with elevated LCN2 also showed a correlation between inflammasome activation and increased fluorescence intensity of the Liperfluo dye, indicative of oxidative stress-induced ferroptosis. Interestingly, both in human AMD patients and in mouse models with a dry AMD-like phenotype (cryba1 cKO and KO), the LCN2 homodimer variant is increased significantly compared to the monomer. Sub-retinal injection of the LCN2 homodimer secreted by RPE cells into NOD-SCID mice leads to retinal degeneration. In addition, we generated an LCN2 monoclonal antibody that neutralizes both the monomer and homodimer variants and rescued autophagy and ferroptosis activities in cryba1 cKO mice. Furthermore, the antibody rescued retinal function in cryba1 cKO mice as assessed by electroretinography. Here, we identify a molecular pathway whereby increased LCN2 elicits pathophysiology in the RPE, cells known to drive dry AMD pathology, thus providing a possible therapeutic strategy for a disease with no current treatment options.Abbreviations: ACTB: actin, beta; Ad-GFP: adenovirus-green fluorescent protein; Ad-LCN2: adenovirus-lipocalin 2; Ad-LCN2-GFP: adenovirus-LCN2-green fluorescent protein; LCN2AKT2: AKT serine/threonine kinase 2; AMBRA1: autophagy and beclin 1 regulator 1; AMD: age-related macular degeneration; ARPE19: adult retinal pigment epithelial cell line-19; Asp278: aspartate 278; ATG4B: autophagy related 4B cysteine peptidase; ATG4C: autophagy related 4C cysteine peptidase; ATG7: autophagy related 7; ATG9B: autophagy related 9B; BLOC-1: biogenesis of lysosomal organelles complex 1; BLOC1S1: biogenesis of lysosomal organelles complex 1 subunit 1; C57BL/6J: C57 black 6J; CGAS: cyclic GMP-AMP synthase; ChQ: chloroquine; cKO: conditional knockout; Cys74: cysteine 74; Dab2: DAB adaptor protein 2; Def: deferoxamine; DHE: dihydroethidium; DMSO: dimethyl sulfoxide; ERG: electroretinography; FAC: ferric ammonium citrate; Fe2+: ferrous; FTH1: ferritin heavy chain 1; GPX: glutathione peroxidase; GST: glutathione S-transferase; H2O2: hydrogen peroxide; His280: histidine 280; IFNL/IFNλ: interferon lambda; IL1B/IL-1ß: interleukin 1 beta; IS: Inner segment; ITGB1/integrin ß1: integrin subunit beta 1; KO: knockout; LC3-GST: microtubule associated protein 1 light chain 3-GST; C-terminal fusion; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LCN2: lipocalin 2; mAb: monoclonal antibody; MDA: malondialdehyde; MMP9: matrix metallopeptidase 9; NLRP3: NLR family pyrin domain containing 3; NOD-SCID: nonobese diabetic-severe combined immunodeficiency; OS: outer segment; PBS: phosphate-buffered saline; PMEL/PMEL17: premelanosome protein; RFP: red fluorescent protein; rLCN2: recombinant LCN2; ROS: reactive oxygen species; RPE SM: retinal pigmented epithelium spent medium; RPE: retinal pigment epithelium; RSL3: RAS-selective lethal; scRNAseq: single-cell ribonucleic acid sequencing; SD-OCT: spectral domain optical coherence tomography; shRNA: small hairpin ribonucleic acid; SM: spent medium; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TYR: tyrosinase; VCL: vinculin; WT: wild type.

The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in atrophic AMD.

Age-related macular degeneration (AMD), the leading cause of geriatric blindness, is a multi-factorial disease with retinal-pigmented epithelial (RPE) cell dysfunction as a central pathogenic driver. With RPE degeneration, lysosomal function is a core process that is disrupted. Transcription factors EB/E3 (TFEB/E3) tightly control lysosomal function; their disruption can cause aging disorders, such as AMD. Here, we show that induced pluripotent stem cells (iPSC)-derived RPE cells with the complement factor H variant [ CFH (Y402H)] have increased AKT2, which impairs TFEB/TFE3 nuclear translocation and lysosomal function. Increased AKT2 can inhibit PGC1α, which downregulates SIRT5, an AKT2 binding partner. SIRT5 and AKT2 co-regulate each other, thereby modulating TFEB-dependent lysosomal function in the RPE. Failure of the AKT2/SIRT5/TFEB pathway in the RPE induced abnormalities in the autophagy-lysosome cellular axis by upregulating secretory autophagy, thereby releasing a plethora of factors that likely contribute to drusen formation, a hallmark of AMD. Finally, overexpressing AKT2 in RPE cells in mice led to an AMD-like phenotype. Thus, targeting the AKT2/SIRT5/TFEB pathway could be a potential therapy for atrophic AMD.

Activated cGAS/STING signaling elicits endothelial cell senescence in early diabetic retinopathy.

Diabetic retinopathy (DR) is a leading cause of blindness in working-age adults and remains an important public health issue worldwide. Here we demonstrate that the expression of stimulator of interferon genes (STING) is increased in patients with DR and animal models of diabetic eye disease. STING has been previously shown to regulate cell senescence and inflammation, key contributors to the development and progression of DR. To investigate the mechanism whereby STING contributes to the pathogenesis of DR, diabetes was induced in STING-KO mice and STINGGT (loss-of-function mutation) mice, and molecular alterations and pathological changes in the retina were characterized. We report that retinal endothelial cell senescence, inflammation, and capillary degeneration were all inhibited in STING-KO diabetic mice; these observations were independently corroborated in STINGGT mice. These protective effects resulted from the reduction in TBK1, IRF3, and NF-κB phosphorylation in the absence of STING. Collectively, our results suggest that targeting STING may be an effective therapy for the early prevention and treatment of DR.