eIF2α incites photoreceptor cell and retina damage by all-trans-retinal.

Dry age-related macular degeneration (AMD) and recessive Stargardt's disease (STGD1) lead to irreversible blindness in humans. The accumulation of all-trans-retinal (atRAL) induced by chaos in visual cycle is closely associated with retinal atrophy in dry AMD and STGD1 but its critical downstream signaling molecules remain ambiguous. Here, we reported that activation of eukaryotic translation initiation factor 2α (eIF2α) by atRAL promoted retinal degeneration and photoreceptor loss through activating c-Jun N-terminal kinase (JNK) signaling-dependent apoptosis and gasdermin E (GSDME)-mediated pyroptosis. We determined that eIF2α activation by atRAL in photoreceptor cells resulted from endoplasmic reticulum homeostasis disruption caused at least in part by reactive oxygen species production, and it activated JNK signaling independent of and dependent on activating transcription factor 4 and the activating transcription factor 4/transcription factor C/EBP homologous protein (CHOP) axis. CHOP overexpression induced apoptosis of atRAL-loaded photoreceptor cells through activating JNK signaling rather than inhibiting the expression of antiapoptotic gene Bcl2. JNK activation by eIF2α facilitated photoreceptor cell apoptosis caused by atRAL via caspase-3 activation and DNA damage. Additionally, we demonstrated that eIF2α was activated in neural retina of light-exposed Abca4-/-Rdh8-/- mice, a model that shows severe defects in atRAL clearance and displays primary features of human dry AMD and STGD1. Of note, inhibition of eIF2α activation by salubrinal effectively ameliorated retinal degeneration and photoreceptor apoptosis in Abca4-/-Rdh8-/- mice upon light exposure. The results of this study suggest that eIF2α is an important target to develop drug therapies for the treatment of dry AMD and STGD1.

Gasdermin E mediates photoreceptor damage by all-trans-retinal in the mouse retina.

The breakdown of all-trans-retinal (atRAL) clearance is closely associated with photoreceptor cell death in dry age-related macular degeneration (AMD) and autosomal recessive Stargardt's disease (STGD1), but its mechanisms remain elusive. Here, we demonstrate that activation of gasdermin E (GSDME) but not gasdermin D promotes atRAL-induced photoreceptor damage by activating pyroptosis and aggravating apoptosis through a mitochondria-mediated caspase-3-dependent signaling pathway. Activation of c-Jun N-terminal kinase was identified as one of the major causes of mitochondrial membrane rupture in atRAL-loaded photoreceptor cells, resulting in the release of cytochrome c from mitochondria to the cytosol, where it stimulated caspase-3 activation required for cleavage of GSDME. Aggregation of the N-terminal fragment of GSDME in the mitochondria revealed that GSDME was likely to penetrate mitochondrial membranes in photoreceptor cells after atRAL exposure. ABC (subfamily A, member 4) and all-trans-retinol dehydrogenase 8 are two key proteins responsible for clearing atRAL in the retina. Abca4-/-Rdh8-/- mice exhibit serious defects in atRAL clearance upon light exposure and serve as an acute model for dry AMD and STGD1. We found that N-terminal fragment of GSDME was distinctly localized in the photoreceptor outer nuclear layer of light-exposed Abca4-/-Rdh8-/- mice. Of note, degeneration and caspase-3 activation in photoreceptors were significantly alleviated in Abca4-/-Rdh8-/-Gsdme-/- mice after exposure to light. The results of this study indicate that GSDME is a common causative factor of photoreceptor pyroptosis and apoptosis arising from atRAL overload, suggesting that repressing GSDME may represent a potential treatment of photoreceptor atrophy in dry AMD and STGD1.

Repressing c-Jun N-terminal kinase signaling mitigates retinal pigment epithelium degeneration in mice with failure to clear all-trans-retinal.

Retinal pigment epithelium (RPE) cell apoptosis arising from all-trans-retinal (atRAL) is in close contact with the etiology of dry age-related macular degeneration (AMD) and autosomal recessive Stargardt's disease (STGD1), but its underlying mechanisms remain elusive. In this study, we reported that c-Jun N-terminal kinase (JNK) activation facilitated atRAL-induced apoptosis of RPE cells. Reactive oxygen species production and endoplasmic reticulum stress were identified as two of major upstream events responsible for activating JNK signaling in atRAL-loaded RPE cells. Inhibiting JNK signaling rescued RPE cells from apoptosis induced by atRAL through attenuating caspase-3 activation leading to poly-ADP-ribose polymerase (PARP) cleavage, and DNA damage response. Abca4-/-Rdh8-/- mice upon light exposure exhibit rapidly increased accumulation of atRAL in the retina, and display severe RPE degeneration, a primary attribute of dry AMD and STGD1. Reducing JNK signaling by intraperitoneally injected JNK-IN-8 was highly effective in preventing RPE atrophy and apoptosis in light-exposed Abca4-/-Rdh8-/- mice. These findings afford a further understanding for contribution of JNK activation by atRAL to retinal damage.

IL-1/IL-1R signaling induced by all-trans-retinal contributes to complement alternative pathway activation in retinal pigment epithelium.

The underlying mechanisms of complement activation in Stargardt disease type 1 (STGD1) and age-related macular degeneration (AMD) are not fully understood. Overaccumulation of all-trans-retinal (atRAL) has been proposed as the pathogenic factor in both diseases. By incubating retinal pigment epithelium (RPE) cells with atRAL, we showed that C5b-9 membrane attack complexes (MACs) were generated mainly through complement alternative pathway. An increase in complement factor B (CFB) expression as well as downregulation of complement regulatory proteins CD46, CD55, CD59, and CFH were observed in RPE cells after atRAL treatment. Furthermore, interleukin-1ß production was provoked in both atRAL-treated RPE cells and microglia/macrophages. Coincubation of RPE cells with interleukin-1 receptor antagonist (IL1Ra) and atRAL ameliorated complement activation and downregulated CFB expression by attenuating both p38 and c-Jun N-terminal kinase (JNK) signaling pathways. Our findings demonstrate that atRAL induces an autocrine/paracrine IL-1/IL-1R signaling to promote complement alternative pathway activation in RPE cells and provide a novel perspective on the pathomechanism of macular degeneration.

Conversion of all-trans-retinal into all-trans-retinal dimer reflects an alternative metabolic/antidotal pathway of all-trans-retinal in the retina.

Free all-trans-retinal (atRAL) and retinal pigment epithelium (RPE) lipofuscin are both considered to play etiological roles in Stargardt disease and age-related macular degeneration. A2E and all-trans-retinal dimer (atRAL-dimer) are two well characterized bisretinoid constituents of RPE lipofuscin. In this study, we found that, after treatment of primary porcine RPE (pRPE) cells with atRAL, atRAL-dimer readily formed and accumulated in a concentration- and time-dependent manner, but A2E was barely detected. Cell-based assays revealed that atRAL, the precursor of atRAL-dimer, significantly altered the morphology of primary pRPE cells and decreased cell viability at a concentration of 80 µm regardless of light exposure. By contrast, atRAL-dimer was not cytotoxic and phototoxic to primary pRPE cells. Compared with atRAL and A2E, atRAL-dimer was more vulnerable to light, followed by the generation of its photocleaved products. Moreover, we observed the presence of atRAL-dimer in reaction mixtures of atRAL with porcine rod outer segments (ROS), RPE/choroid, or neural retina. Taken together, we here proposed an alternative metabolic/antidotal pathway of atRAL in the retina: atRAL that evades participation of the visual (retinoid) cycle undergoes a condensation reaction to yield atRAL-dimer in both ROS and RPE. Translocation of atRAL, all-trans N-retinylidene-phosphatidylethanolamine (NR-PE), atRAL-dimer, and photocleavage products of atRAL-dimer from ROS into RPE is accomplished by phagocytosing shed ROS on a daily basis. Without causing damage to RPE cells, light breaks up total atRAL-dimer within RPE cells to release low-molecular-weight photocleavage fragments. The latter, together with ROS-atRAL-dimer photocleavage products, may easily move across membranes and thereby be metabolically eliminated.

Induction of ferroptosis by HO-1 contributes to retinal degeneration in mice with defective clearance of all-trans-retinal.

The accumulation of all-trans-retinal (atRAL) in photoreceptors and the retinal pigment epithelium (RPE), which is induced by chaos in visual (retinoid) cycle, is closely associated with the pathogenesis of dry age-related macular degeneration (AMD) and autosomal recessive Stargardt's disease (STGD1). Although we have reported that the induction of ferroptosis by atRAL is an important cause of photoreceptor loss, but its mechanisms still remain unclear. In this study, we identified heme oxygenase-1 (HO-1) as an inducer of photoreceptor ferroptosis elicited by atRAL. In atRAL-loaded photoreceptor cells, inhibition of Kelch-like ECH-associated protein 1 (KEAP1) at least in part by reactive oxygen species (ROS) production evoked the release of nuclear factor-erythroid 2-related factor-2 (NRF2) from KEAP1, followed by the translocation of active NRF2 into the nucleus where it promoted the transcription of the Ho-1 gene, thereby leading to HO-1 overexpression in the cytosol. A significant elevation of Fe2+ levels in photoreceptor cells resulted from activation of HO-1 by atRAL, and it facilitated ROS overproduction and then triggered ferroptotic cell death through ROS-mediated lipid peroxidation. Both treatment with HO-1 repressor Zinc protoporphyrin IX (ZnPP) and knockout of Ho-1 gene clearly rescued photoreceptor cells against ferroptosis arising from atRAL overload. Light-exposed Abca4-/-Rdh8-/- mice rapidly display severe defects in atRAL clearance, and serve as an acute model of dry AMD and STGD1. HO-1 activation was distinctly observed in neural retina of Abca4-/-Rdh8-/- mice after exposure to light, and it was visibly relieved by intraperitoneally injected ferroptosis inhibitor ferrostatin-1. More notably, intraperitoneal administration of ZnPP effectively alleviated both photoreceptor degeneration and RPE atrophy in Abca4-/-Rdh8-/- mice in response to light exposure by repressing HO-1-mediated ferroptosis. Our study suggests that HO-1 is a key factor that regulates atRAL-induced ferroptosis in photoreceptors and the RPE, and its inhibition may hold promises for the therapy of dry AMD and STGD1.

Effect of palmitoylethanolamide on degeneration of a human-derived retinal pigment epithelial cell induced by all-trans retinal.

AIM: To study the effect of palmitoylethanolamide (PEA) on apoptosis of retinal pigment epithelial (RPE) cells induced by all-trans retinal (atRAL) and to explore the possible molecular mechanism. METHODS: CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS) was used to detect the effect of PEA on human-derived retinal epithelial cells (ARPE-19) viability induced by atRAL. A Leica DMi8 inverted microscope was used to observe cell morphology. Reactive oxygen species (ROS) production was evaluated with 2',7'-dichlorodihydrof-luorescein diacetate (H2DCFDA) staining and fluorescence microscopy. Expression of c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), c-Jun, phosphorylated c-Jun (p-c-Jun), Bak, cleaved caspase-3, C/EBP homologous protein (CHOP), and binding (Bip) protein levels were tested by Western blot. Abca4 -/- Rdh8 -/- mice, mouse models of atRAL clearance defects which displays some symbolic characteristics of dry age-related macular degeneration (AMD) and Stargardt disease (STGD1). In the animal models, PEA was injected intraperitoneally. The full-field electroretinogram was used to detect visual function under scotopic conditions traced from mice. Optical coherence tomography showed reconstitution or thickening of the retinal pigment epithelium layer. Effect of PEA on fundus injury induced by light in Abca4-/-Rdh8-/- mice was observed by fundus photography. RESULTS: PEA ameliorated ARPE-19 cells apoptosis and inhibited ROS (including mitochondrial ROS) production induced by atRAL. PEA improved the retinal functional, prohibited both RPE and photoreceptor from death, ameliorates light-induced fundus impairment in Abca4 -/- Rdh8 -/- mice. In vitro and in vivo, PEA inhibited JNK, p-JNK, c-Jun, p-c-Jun, Bak, cleaved caspase-3, CHOP, and Bip protein levels induced by all-trans retinal in ARPE-19 cells. CONCLUSION: PEA has effect on treating RPE cells apoptosis in retinopathy caused by atRAL accumulation. PEA is a potential treatment strategy for dry AMD and STGD1. The molecular mechanism is affecting the ROS-JNK-CHOP signaling pathway partly.

Primary Culture of Porcine Retinal Pigment Epithelial Cells.

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Cyanidin-3-glucoside improves the barrier function of retinal pigment epithelium cells by attenuating endoplasmic reticulum stress-induced apoptosis.

Excessive exposure to blue light from smartphones, computers, and other video equipment causes retinal degeneration. Cyanidin-3-glucoside (C3G) exerts protective effects on retinal cells. However, the mechanism by which C3G enhances the barrier function of retinal pigment epithelial (RPE) cells remains unclear. This study investigated the effects of C3G on blue light-irradiated A2E-containing RPE cells and explored whether or not the endoplasmic reticulum (ER) stress and downstream nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways are involved in the mechanism. Results showed that C3G (10 and 25 µM) observably increased the viability and inhibited the apoptosis of RPE cells. Furthermore, C3G enhanced the barrier function of RPE cells and upregulated the expression of tight junction proteins. Blue light irradiation triggered ER stress, but C3G significantly suppressed the PERK/eIF2α/ATF4/CHOP pathway and maintained normal ER morphology in RPE cells. C3G also activated the Nrf2 pathway to promote RPE survival, which was independent of ER stress modulating Nrf2 activity. This study suggests that C3G promotes the barrier function of RPE cells by regulating ER stress-induced apoptosis, thereby offering a new approach to preventing retinal diseases. Thus, C3G is a potential functional food ingredient to improve visual health.

Retinal Pigment Epithelium Cell Death Is Associated With NLRP3 Inflammasome Activation by All-trans Retinal.

Purpose: Visual (retinoid) cycle anomalies induce aberrant build-up of all-trans retinal (atRAL) in the retinal pigment epithelium (RPE), which is a cause of RPE atrophy in Stargardt disease type 1 and age-related macular degeneration. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation is implicated in the etiology of age-related macular degeneration. Here, we elucidated the relationship between NLRP3 inflammasome activation and atRAL-induced death of RPE cells. Methods: Cellular toxicities were assessed by MTS or MTT assays. Expression levels of mRNAs and proteins were determined by quantitative reverse transcription-polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. Fluorescence microscopy was used to examine intracellular signals. Ultrastructural features of organelles were examined by transmission electron microscope. Results: Abnormal accumulation of atRAL was associated with a significant increase in the proportion of human ARPE-19 cells exhibiting features of apoptosis and Caspase-3/gasdermin E (GSDME)-mediated pyroptosis. These cells also exhibited elevated expression of NLRP3, ASC, cleaved Caspase-1/poly ADP-ribose polymerase (PARP)/Caspase-3/GSDME, interleukin-1ß (IL-1ß), and IL-18, as well as NLRP3 inflammasome-related genes (IL1B and IL18). After exposure of human ARPE-19 cells to excess atRAL, reactive oxygen species (ROS) (including mitochondrial ROS) and cathepsins released from lysosomes transmitted signals leading to NLRP3 inflammasome activation. Suppressing the production of ROS, NLRP3 inflammasome, Caspase-1, cathepsin B, or cathepsin D protected ARPE-19 cells against atRAL-associated cytotoxicity. Damage to mitochondria, lysosomes, and endoplasmic reticulum in atRAL-exposed ARPE-19 cells was partially alleviated by treatment with MCC950, a selective NLRP3 inflammasome inhibitor. Conclusions: Aberrant build-up of atRAL promotes the death of RPE cells via NLRP3 inflammasome activation.