RAR Inhibitors Display Photo-Protective and Anti-Inflammatory Effects in A2E Stimulated RPE Cells In Vitro through Non-Specific Modulation of PPAR or RXR Transactivation.

N-retinylidene-N-retinylethanolamine (A2E) has been associated with age-related macular degeneration (AMD) physiopathology by inducing cell death, angiogenesis and inflammation in retinal pigmented epithelial (RPE) cells. It was previously thought that the A2E effects were solely mediated via the retinoic acid receptor (RAR)-α activation. However, this conclusion was based on experiments using the RAR "specific" antagonist RO-41-5253, which was found to also be a ligand and partial agonist of the peroxisome proliferator-activated receptor (PPAR)-γ. Moreover, we previously reported that inhibiting PPAR and retinoid X receptor (RXR) transactivation with norbixin also modulated inflammation and angiogenesis in RPE cells challenged in the presence of A2E. Here, using several RAR inhibitors, we deciphered the respective roles of RAR, PPAR and RXR transactivations in an in vitro model of AMD. We showed that BMS 195614 (a selective RAR-α antagonist) displayed photoprotective properties against toxic blue light exposure in the presence of A2E. BMS 195614 also significantly reduced the AP-1 transactivation and mRNA expression of the inflammatory interleukin (IL)-6 and vascular endothelial growth factor (VEGF) induced by A2E in RPE cells in vitro, suggesting a major role of RAR in these processes. Surprisingly, however, we showed that (1) Norbixin increased the RAR transactivation and (2) AGN 193109 (a high affinity pan-RAR antagonist) and BMS 493 (a pan-RAR inverse agonist), which are photoprotective against toxic blue light exposure in the presence of A2E, also inhibited PPARs transactivation and RXR transactivation, respectively. Therefore, in our in vitro model of AMD, several commercialized RAR inhibitors appear to be non-specific, and we propose that the phototoxicity and expression of IL-6 and VEGF induced by A2E in RPE cells operates through the activation of PPAR or RXR rather than by RAR transactivation.

In Memory of Anthony Stevens: A Career Retrospective with Emphasis on His Formative Role in the Archetype Debate.

This paper celebrates the life and legacy of psychiatrist and Jungian author Anthony Stevens, who passed away at age 90 on July 13, 2023. It outlines Stevens's origins as a research fellow in Greece, where his work on infant attachment led to a lifelong dedication to establishing the biological and evolutionary foundation of psychiatry. It details his instrumental role in the debate about the theory of archetypes and describes the current state of the literature including the responses and reactions to Stevens's biological innatist position. The paper concludes with a career retrospective in which Stevens's major works are introduced and briefly described.

Cet article célèbre la vie et l'héritage du psychiatre et auteur jungien Anthony Stevens, décédé à l'âge de 90 ans le 13 juillet 2023. L'article décrit les origines de Stevens en tant que chercheur en Grèce, où ses travaux sur l'attachement du nourrisson l'ont conduit à se consacrer toute sa vie à établir le fondement biologique de la psychiatrie, dans une perspective évolutionniste. L'article détaille son rôle important dans le débat sur la théorie des archétypes et décrit l'état actuel de la littérature, y compris les réponses et réactions à la position biologique innéiste de Stevens. L'article se termine par une rétrospective de sa carrière, dans laquelle les œuvres majeures de Stevens sont présentées et brièvement décrites.

Este artículo celebra la vida y el legado del psiquiatra y autor Junguiano Anthony Stevens, quien falleció a los 90 años el 13 de julio de 2023. Describe los orígenes de Stevens como investigador en Grecia, donde su trabajo sobre el apego infantil lo llevó a una dedicación de por vida para establecer los fundamentos biológicos y evolutivos de la psiquiatría. Detalla su papel instrumental en el debate sobre la teoría de los arquetipos y describe el estado actual del arte, incluyendo las respuestas y reacciones a la posición biológica innatista de Stevens. El artículo concluye con una retrospectiva de su carrera en la que se presentan y describen brevemente las principales obras de Stevens.

N-retinylidene-N-retinylethanolamine degradation in human retinal pigment epithelial cells via memantine- and ifenprodil-mediated autophagy.

N-methyl-D-aspartate (NMDA) receptors are ionic glutamine receptors involved in brain development and functions such as learning and memory formation. NMDA receptor inhibition is associated with autophagy activation. In this study, we investigated whether the NMDA receptor antagonists, memantine and ifenprodil, induce autophagy in human retinal pigment epithelial cells (ARPE-19) to remove Nretinylidene- N-retinylethanolamine (A2E), an intracellular lipofuscin component. Fluorometric analysis using labeled A2E (A2E-BDP) and confocal microscopic examination revealed that low concentrations of NMDA receptor antagonists, which did not induce cytotoxicity, significantly reduced A2E accumulation in ARPE-19 cells. In addition, memantine and ifenprodil activated autophagy in ARPE-19 cells as measured by microtubule-associated protein 1A/1B-light chain3-II formation and phosphorylated p62 protein levels. Further, to understand the correlation between memantine- and ifenprodil-mediated A2E degradation and autophagy, autophagy-related 5 (ATG5) was depleted using RNA interference. Memantine and ifenprodil failed to degrade A2E in ARPE-19 cells lacking ATG5. Taken together, our study indicates that the NMDA receptor antagonists, memantine and ifenprodil, can remove A2E accumulated in cells via autophagy activation in ARPE-19 cells.

Macular Pigment Carotenoids and Bisretinoid A2E.

Lutein (L), zeaxanthin (Z), and meso-zeaxanthin (MZ) are the three macular pigments (MP) carotenoids that uniquely accumulate in the macula lutea region of the human retina. L and Z are obtained by humans through dietary intake. The third MP, MZ, is rarely present in diet, and its abundance in the human fovea is due to the metabolic conversion of dietary L by the retinal pigment epithelium's RPE65 enzyme. The major functions of MP in ocular health are to filter high-intensity, phototoxic blue light and to act as effective antioxidants for scavenging free radicals. The pyridinium bisretinoid, N-retinylidene-N-retinylethanolamine (A2E), contributes to drusen formation in dry age-related macular degeneration (AMD) and to the autofluorescent flecks in autosomal recessive Stargardt disease (STGD1). Retinal carotenoids attenuate A2E formation and can directly and indirectly alleviate A2E-mediated oxidative damage. In this chapter, we review these more recently recognized interconnections between MP carotenoids and A2E bisretinoids.

Anti-Inflammatory Effects and Photo- and Neuro-Protective Properties of BIO203, a New Amide Conjugate of Norbixin, in Development for the Treatment of Age-Related Macular Degeneration (AMD).

9'-cis-norbixin (norbixin/BIO201) protects RPE cells against phototoxicity induced by blue light and N-retinylidene-N-retinylethanolamine (A2E) in vitro and preserves visual functions in animal models of age-related macular degeneration (AMD) in vivo. The purpose of this study was to examine the mode of action and the in vitro and in vivo effects of BIO203, a novel norbixin amide conjugate. Compared to norbixin, BIO203 displays improved stability at all temperatures tested for up to 18 months. In vitro, BIO203 and norbixin share a similar mode of action involving the inhibition of PPARs, NF-κB, and AP-1 transactivations. The two compounds also reduce IL-6, IL-8, and VEGF expression induced by A2E. In vivo, ocular maximal concentration and BIO203 plasma exposure are increased compared to those of norbixin. Moreover, BIO203 administered systemically protects visual functions and retinal structure in albino rats subjected to blue-light illumination and in the retinal degeneration model of Abca4-/- Rdh8-/- double knock-out mice following 6 months of oral complementation. In conclusion, we report here that BIO203 and norbixin share similar modes of action and protective effects in vitro and in vivo. BIO203, with its improved pharmacokinetic and stability properties, could be developed for the treatment of retinal degenerative diseases such as AMD.

Protective Effects of Dipterocarpus tuberculatus in Blue Light-Induced Macular Degeneration in A2E-Laden ARPE19 Cells and Retina of Balb/c Mice.

Natural products with significant antioxidant activity have been receiving attention as one of the treatment strategies to prevent age-related macular degeneration (AMD). Reactive oxygen intermediates (ROI) including oxo-N-retinylidene-N-retinylethanolamine (oxo-A2E) and singlet oxygen-induced damage, are believed to be one of the major causes of the development of AMD. To investigate the therapeutic effects of methanol extracts of Dipterocarpus tuberculatus Roxb. (MED) against blue light (BL)-caused macular degeneration, alterations in the antioxidant activity, apoptosis pathway, neovascularization, inflammatory response, and retinal degeneration were analyzed in A2E-laden ARPE19 cells and Balb/c mice after exposure of BL. Seven bioactive components, including 2α-hydroxyursolic acid, ε-viniferin, asiatic acid, bergenin, ellagic acid, gallic acid and oleanolic acid, were detected in MED. MED exhibited high DPPH and ABTS free radical scavenging activity. BL-induced increases in intracellular reactive oxygen species (ROS) production and nitric oxide (NO) concentration were suppressed by MED treatment. A significant recovery of antioxidant capacity by an increase in superoxide dismutase enzyme (SOD) activity, SOD expression levels, and nuclear factor erythroid 2-related factor 2 (NRF2) expression were detected as results of MED treatment effects. The activation of the apoptosis pathway, the expression of neovascular proteins, cyclooxygenase-2 (COX-2)-induced inducible nitric oxide synthase (iNOS) mediated pathway, inflammasome activation, and expression of inflammatory cytokines was remarkably inhibited in the MED treated group compared to the Vehicle-treated group in the AMD cell model. Furthermore, MED displayed protective effects in BL-induced retinal degeneration through improvement in the thickness of the whole retina, outer nuclear layer (ONL), inner nuclear layer (INL), and photoreceptor layer (PL) in Balb/c mice. Taken together, these results indicate that MED exhibits protective effects in BL-induced retinal degeneration and has the potential in the future to be developed as a treatment option for dry AMD with atrophy of retinal pigment epithelial (RPE) cells.

Protective effects of Panax ginseng berry extract on blue light-induced retinal damage in ARPE-19 cells and mouse retina.

Background: Age-related macular degeneration (AMD) is a significant visual disease that induces impaired vision and irreversible blindness in the elderly. However, the effects of ginseng berry extract (GBE) on the retina have not been studied. Therefore, this study aimed to investigate the protective effects of GBE on blue light (BL)-induced retinal damage and elucidate its underlying mechanisms in human retinal pigment epithelial cells (ARPE-19 cells) and Balb/c retina. Methods: To investigate the effects and underlying mechanisms of GBE on retinal damage in vitro, we performed cell viability assay, pre-and post-treatment of sample, reactive oxygen species (ROS) assay, quantitative real-time PCR (qRT-PCR), and western immunoblotting using A2E-laden ARPE-19 cells with BL exposure. In addition, Balb/c mice were irradiated with BL to induce retinal degeneration and orally administrated with GBE (50, 100, 200 mg/kg). Using the harvested retina, we performed histological analysis (thickness of retinal layers), qRT-PCR, and western immunoblotting to elucidate the effects and mechanisms of GBE against retinal damage in vivo. Results: GBE significantly inhibited BL-induced cell damage in ARPE-19 cells by activating the SIRT1/PGC-1α pathway, regulating NF-kB translocation, caspase 3 activation, PARP cleavage, expressions of apoptosis-related factors (BAX/BCL-2, LC3-Ⅱ, and p62), and ROS production. Furthermore, GBE prevented BL-induced retinal degeneration by restoring the thickness of retinal layers and suppressed inflammation and apoptosis via regulation of NF-kB and SIRT1/PGC-1α pathway, cleavage of caspase 3 and PARP, and expressions of apoptosis-related factors in vivo. Conclusions: GBE could be a potential agent to prevent dry AMD and progression to wet AMD.

Efficacy of created and restored nesting sites for the conservation of colonial Laridae in the South of France.

By rapidly modifying key habitat components, habitat restoration is at risk of producing attractive cues for animals without providing habitats of sufficient quality. As such, individual fitness components, such as reproduction, could be reduced and restored habitats could become ecological traps. This risk notably appears by using artificial constructions in restoration projects, yet few studies have evaluated their efficacy in a robust way. We investigated this by analyzing 154 islets that were created or restored to improve the conservation status of 7 colonial Laridae species in the South of France. From 2007 to 2016, we compared occupancy dynamics and breeding parameters of these species between the restored sites and 846 unmanaged nesting sites. We also explored species' preference for different nesting site characteristics and their respective effect on breeding parameters. Restored nesting sites were 2-9 times as attractive as unmanaged sites for all species except the Black-headed Gull (Chroicocephalus ridibundus). Colonization probability was up to 100 times higher in sites already used by other species the previous year and increased with distance to the shore until >0.2 when distance was over 250 m. Abandonment probability was 29-70% lower when breeding was successful the previous year in all species except the Sandwich Tern (Thalasseus sandvicensis). Productivity and breeding success probability were 2 times higher on managed sites. Distance from the shore was an important attractive characteristic of artificial nesting sites in all species. Other nesting site characteristics had species-specific effects on colonization, abandonment, and breeding success. Our results indicate that managed nesting sites are successful conservation tools for colonial Laridae in the Mediterranean and do not act as ecological traps. Our study showed that testing the ecological trap hypothesis is a robust way to evaluate the success of restoration projects of breeding habitats.

Eficiencia de los sitios de anidación creados y restaurados para la conservación de láridos coloniales en el sur de Francia Resumen Con la rápida modificación de los componentes clave de un hábitat, la restauración corre el riesgo de producir entradas atractivas para los animales sin proporcionar hábitats con la suficiente calidad. Como tal, los elementos individuales de la aptitud, como la reproducción, podrían ser reducidos y los hábitats restaurados podrían convertirse en trampas ecológicas. Aunque este riesgo aparece especialmente cuando se usan construcciones artificiales en los proyectos de restauración, son pocos los estudios que han evaluado su efectividad de manera firme. Investigamos lo anterior con el análisis de 154 islotes que fueron creados o restaurados para mejorar el estado de conservación de siete especies de láridos coloniales en el sur de Francia. Comparamos las dinámicas de ocupación y los parámetros de reproducción de estas especies entre 2007 y 2016 en los sitios restaurados y en 846 sitios de anidación no administrados. También exploramos la preferencia de las especies por diferentes características en los sitios de anidación y su respectivo efecto sobre los parámetros de reproducción. Los sitios de anidación restaurados fueron de 2 a 9 veces más atractivos para todas las especies, excepto la gaviota de cabeza negra (Croicocephalus ridibundus), que los sitios no administrados. La probabilidad de colonización fue hasta 100 veces mayor en los sitios usados por otras especies el año previo e incrementó con la distancia a la costa hasta >0.2, cuando la distancia fue mayor a los 250 metros. La probabilidad de abandono fue de 29 a 70% más baja para todas las especies, excepto el charrán de Sándwich (Thalasseus sandvicensis), cuando la reproducción fue exitosa el año anterior. La probabilidad de la productividad y el éxito de reproducción fueron dos veces mayores en los sitios administrados. La distancia a la costa fue una característica atractiva importante de los sitios artificiales de anidación para todas las especies. Otras características de los sitios de anidación tuvieron efectos específicos por especie sobre la colonización, el abandono y el éxito de la reproducción. Nuestros resultados indican que los sitios de anidación administrados son herramientas exitosas de conservación para los láridos coloniales en el Mediterráneo y no funcionan como trampas ecológicas. Nuestro estudio demuestra que analizar la hipótesis de la trampa ecológica es una manera sólida de evaluar el éxito de la restauración en los proyectos de hábitats para la reproducción.

Epitranscriptome Analysis of Oxidative Stressed Retinal Epithelial Cells Depicted a Possible RNA Editing Landscape of Retinal Degeneration.

Oxidative stress represents one of the principal causes of inherited retinal dystrophies, with many related molecular mechanisms still unknown. We investigated the posttranscriptional RNA editing landscape of human retinal pigment epithelium cells (RPE) exposed to the oxidant agent N-retinylidene-N-retinyl ethanolamine (A2E) for 1 h, 2 h, 3 h and 6 h. Using a transcriptomic approach, refined with a specific multialgorithm pipeline, 62,880 already annotated and de novo RNA editing sites within about 3000 genes were identified among all samples. Approximately 19% of these RNA editing sites were found within 3' UTR, including sites common to all time points that were predicted to change the binding capacity of 359 miRNAs towards 9654 target genes. A2E exposure also determined significant gene expression differences in deaminase family ADAR, APOBEC and ADAT members, involved in canonical and tRNA editing events. On GO and KEGG enrichment analyses, genes that showed different RNA editing levels are mainly involved in pathways strongly linked to a possible neovascularization of retinal tissue, with induced apoptosis mediated by the ECM and surface protein altered signaling. Collectively, this work demonstrated dynamic RNA editome profiles in RPE cells for the first time and shed more light on new mechanisms at the basis of retinal degeneration.

NMDA Receptor Antagonists Degrade Lipofuscin via Autophagy in Human Retinal Pigment Epithelial Cells.

Background and Objectives: Age-related macular degeneration is a slow-progressing disease in which lipofuscin accumulates in the retina, causing inflammation and apoptosis of retinal pigment epithelial (RPE) cells. This study aimed to identify N-methyl-D-aspartate (NMDA) signaling as a novel mechanism for scavenging N-retinylidene-N-retinylethanolamine (A2E), a component of ocular lipofuscin, in human RPE cells. Materials and Methods: A2E degradation assays were performed in ARPE-19 cells using fluorescently labeled A2E. The autophagic activity in ARPE-19 cells was measured upon blue light (BL) exposure, after A2E treatment. Autophagy flux was determined by measuring LC3-II formation using immunoblotting and confocal microscopy. To determine whether autophagy via the NMDA receptor is involved in A2E clearance, ATG5-deficient cells were used. Results: Ro 25-6981, an NR2B-selective NMDA receptor antagonist, effectively cleared A2E. Ro 25-6981 reduced A2E accumulation in the lysosomes of ARPE-19 cells at sub-cytotoxic concentrations, while increasing the formation of LC3-II and decreasing p62 protein levels in a concentration-dependent manner. The autophagic flux monitored by RFP-GFP-LC3 and bafilomycin A1 assays was significantly increased by Ro 25-6981. A2E clearance by Ro 25-6981 was abolished in ATG5-depleted ARPE-19 cells, suggesting that A2E degradation by Ro 25-6981 was mediated by autophagy. Furthermore, treatment with other NMDA receptor antagonists, CP-101,606 and AZD6765, showed similar effects on autophagy activation and A2E degradation in ARPE-19 cells. In contrast, glutamate, an NMDA receptor agonist, exhibited a contrasting effect, suggesting that both the activation of autophagy and the degradation of A2E by Ro 25-6981 in ARPE-19 cells occur through inhibition of the NMDA receptor pathway. Conclusions: This study demonstrates that NMDA receptor antagonists degrade lipofuscin via autophagy in human RPE cells and suggests that NMDA receptor antagonists could be promising new therapeutics for retinal degenerative diseases.

The effect of A2E on lysosome membrane permeability during blue light-induced human RPEs apoptosis.

BACKGROUND: To investigate the effect of N-retinyl-N-retinylidene ethanolamine (A2E) on lysosome membrane permeability (LMP) during blue light-induced human retinal pigment epithelium cells (RPEs) apoptosis. METHODS: By building an A2E and blue light irradiation inducing RPEs damage model, the CCK-8 assay was used to detect RPEs viability loaded with different concentrations of A2E after different culturing time to determine the optimum A2E loading concentration. And the RPEs fluorescence intensity changes were observed by fluorescence microscopy loaded with different concentration of A2E. The RPEs were divided into four groups randomly: control group, A2E-loaded group, blue light irradiation group, and A2E-loaded + blue light irradiation group. Annexin V-FITC/PI and TUNEL/DAPI methods were used to detect RPEs apoptotic rate. Laser scanning confocal microscopy (LSCM) was used to observe RPEs LMP changes stained by acridine orange (AO) method. RESULTS: The CCK-8 result showed a downward trend in cells viability of RPEs loaded with increasing concentration of A2E and extending culturing time. The optimum A2E loading concentration was determined at 25 µmol/L. With increasing A2E loading concentrations, the intensity of fluorescence in RPEs decreased gradually. The RPEs apoptotic rate in blue light irradiation + A2E-loaded group was significantly higher than those in other three groups detected by Annexin V-FITC/PI method, which was similar to TUNEL/DAPI's result. After AO staining, cytoplasmic and nucleolar RNAs emits green fluorescence; lysosomes emit red fluorescence. Through the interference of A2E and blue light on RPEs, red fluorescent leakage from the lysosomes (means LMP increasing) can be observed. The mean red fluorescence intensity was chosen as the statistics indicator to estimate LMP change in RPEs cultured in vitro. Compared with the control group, the red fluorescence intensity decreased in A2E-loaded group, blue light irradiation group, and blue light irradiation + A2E-loaded group. Meanwhile, the mean red fluorescence intensity in blue light irradiation + A2E-loaded group was the lowest. CONCLUSIONS: Both A2E-loaded and blue light irradiation could induce human RPEs apoptosis, and the two factors had a synergistic effect. In addition, both A2E and blue light can lead to LMP increasing, which indicated LMP change might be the upstream part in inducing mitochondrion-dependent apoptotic pathway. These data provided evidence that A2E as the most important auto-fluorescence substance in lipofuscin is an initiator of blue light-mediated damage of RPEs and participate in pathogenesis of retinal degenerative diseases in humans.

Protective Effect of Ribes nigrum Extract against Blue Light-Induced Retinal Degeneration In Vitro and In Vivo.

Although blackcurrant has several health benefits, such as antioxidant and anti-inflammatory properties, its effects on the retina remain unclear. In this study, we investigated the efficacy of black currant extract (BCE) in an in vitro and in vivo model of dry age-related macular degeneration (AMD) induced by blue light. Dry macular degeneration is characterized by the abnormal accumulation of lipofuscin (e.g., N-retinylidene-N-retinylethanolamine, A2E) in the retina. Blue light (BL) significantly decreased the viability of A2E-laden human retinal pigment epithelial cells (ARPE-19). However, BCE treatment protected ARPE-19 cells from A2E and BL. A2E, which is oxidized by blue light, generates reactive oxygen species in RPE cells. Treatment with BCE significantly decreased (80.8%) reactive oxygen species levels induced by A2E and BL in a concentration-dependent manner. BCE inhibited A2E accumulation in ARPE-19 cells and significantly downregulated the expression of genes increased by A2E and BL in ARPE-19 cells. In vivo, oral administration of BCE (25-100 mg/kg) ameliorated ocular lesions of BL-induced retinal damage in a mouse model and rescued the thickness of the whole retina, photoreceptor segment layer, outer nuclear layer, and inner nuclear layer. The decrease in the number of nuclei in the outer nuclear layer induced by BL was also rescued by BCE. Additionally, BCE administration rescued (40.0%) the BL-induced reduction in the expression level of superoxide dismutase 1. Taken together, our results suggest that BCE may have preventive and therapeutic effects on dry AMD through its antioxidant activity and inhibition of lipofuscin accumulation in the retina.