RESUMEN
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.
Asunto(s)
Factor 2 Eucariótico de Iniciación , Degeneración Retiniana , Retinaldehído , Enfermedad de Stargardt , Animales , Humanos , Ratones , Factor de Transcripción Activador 4/metabolismo , Apoptosis , Transportadoras de Casetes de Unión a ATP/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Retinaldehído/metabolismo , Enfermedad de Stargardt/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismoRESUMEN
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.
Asunto(s)
Células Fotorreceptoras , Proteínas Citotóxicas Formadoras de Poros , Retina , Retinaldehído , Enfermedad de Stargardt , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Caspasa 3/metabolismo , Ratones , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Retina/metabolismo , Retina/patología , Retinaldehído/metabolismo , Enfermedad de Stargardt/metabolismo , Enfermedad de Stargardt/patologíaRESUMEN
The death of photoreceptor cells in dry age-related macular degeneration (AMD) and autosomal recessive Stargardt disease (STGD1) is closely associated with disruption in all-trans-retinal (atRAL) clearance in neural retina. In this study, we reveal that the overload of atRAL leads to photoreceptor degeneration through activating ferroptosis, a nonapoptotic form of cell death. Ferroptosis of photoreceptor cells induced by atRAL resulted from increased ferrous ion (Fe2+), elevated ACSL4 expression, system Xc- inhibition, and mitochondrial destruction. Fe2+ overload, tripeptide glutathione (GSH) depletion, and damaged mitochondria in photoreceptor cells exposed to atRAL provoked reactive oxygen species (ROS) production, which, together with ACSL4 activation, promoted lipid peroxidation and thereby evoked ferroptotic cell death. Moreover, exposure of photoreceptor cells to atRAL activated COX2, a well-accepted biomarker for ferroptosis onset. In addition to GSH supplement, inhibiting either Fe2+ by deferoxamine mesylate salt (DFO) or lipid peroxidation with ferrostatin-1 (Fer-1) protected photoreceptor cells from ferroptosis caused by atRAL. Abca4-/-Rdh8-/- mice exhibiting defects in atRAL clearance is an animal model for dry AMD and STGD1. We observed that ferroptosis was indeed present in neural retina of Abca4-/-Rdh8-/- mice after light exposure. More importantly, photoreceptor atrophy and ferroptosis in light-exposed Abca4-/-Rdh8-/- mice were effectively alleviated by intraperitoneally injected Fer-1, a selective inhibitor of ferroptosis. Our study suggests that ferroptosis is one of the important pathways of photoreceptor cell death in retinopathies arising from excess atRAL accumulation and should be pursued as a novel target for protection against dry AMD and STGD1.
Asunto(s)
Ferroptosis , Peroxidación de Lípido , Degeneración Macular/patología , Células Fotorreceptoras de Vertebrados/patología , Retinaldehído/análogos & derivados , Animales , Degeneración Macular/metabolismo , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Células Fotorreceptoras de Vertebrados/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Retinaldehído/metabolismo , Enfermedad de Stargardt/metabolismo , Enfermedad de Stargardt/patologíaRESUMEN
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.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Degeneración Retiniana/prevención & control , Epitelio Pigmentado de la Retina/patología , Retinaldehído/metabolismo , Transducción de Señal/fisiología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Animales , Apoptosis , Western Blotting , Caspasa 3/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Estrés del Retículo Endoplásmico/fisiología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especies Reactivas de Oxígeno/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/metabolismo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Disrupted clearance of all-trans-retinal (atRAL), a component of the visual (retinoid) cycle in the retina, may cause photoreceptor atrophy in autosomal recessive Stargardt disease (STGD1) and dry age-related macular degeneration (AMD). However, the mechanisms underlying atRAL-induced photoreceptor loss remain elusive. Here, we report that atRAL activates c-Jun N-terminal kinase (JNK) signaling at least partially through reactive oxygen species production, which promoted mitochondria-mediated caspase- and DNA damage-dependent apoptosis in photoreceptor cells. Damage to mitochondria in atRAL-exposed photoreceptor cells resulted from JNK activation, leading to decreased expression of Bcl2 apoptosis regulator (Bcl2), increased Bcl2 antagonist/killer (Bak) levels, and cytochrome c (Cyt c) release into the cytosol. Cytosolic Cyt c specifically provoked caspase-9 and caspase-3 activation and thereby initiated apoptosis. Phosphorylation of JNK in atRAL-loaded photoreceptor cells induced the appearance of γH2AX, a sensitive marker for DNA damage, and was also associated with apoptosis onset. Suppression of JNK signaling protected photoreceptor cells against atRAL-induced apoptosis. Moreover, photoreceptor cells lacking Jnk1 and Jnk2 genes were more resistant to atRAL-associated cytotoxicity. The Abca4-/-Rdh8-/- mouse model displays defects in atRAL clearance that are characteristic of STGD1 and dry AMD. We found that JNK signaling was activated in the neural retina of light-exposed Abca4-/-Rdh8-/- mice. Of note, intraperitoneal administration of JNK-IN-8, which inhibits JNK signaling, effectively ameliorated photoreceptor degeneration and apoptosis in light-exposed Abca4-/-Rdh8-/- mice. We propose that pharmacological inhibition of JNK signaling may represent a therapeutic strategy for preventing photoreceptor loss in retinopathies arising from atRAL overload.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retinaldehído/farmacología , Transducción de Señal/efectos de los fármacos , Enfermedad de Stargardt/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Animales , Apoptosis/genética , Ratones , Ratones Noqueados , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 9 Activada por Mitógenos/genética , Células Fotorreceptoras de Vertebrados/patología , Transducción de Señal/genética , Enfermedad de Stargardt/genética , Enfermedad de Stargardt/patologíaRESUMEN
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.
Asunto(s)
Epitelio Pigmentado de la Retina/metabolismo , Retinaldehído/análogos & derivados , Retinaldehído/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Hidrólisis , Luz , Redes y Vías Metabólicas , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de la radiación , Segmento Externo de la Célula en Bastón/metabolismo , Porcinos , Espectrometría de Masas en TándemRESUMEN
Toxic lipofuscin in the RPE (retinal pigment epithelium) is implicated in blindness in AMD (age-related macular degeneration) or recessive Stargardt's disease patients. In the present study, we identified a novel fluorescent lipofuscin component in human and bovine RPEs. Using 1D and 2D NMR and MS, we confirmed the structure of this pigment and called it pdA2E. It exhibits absorbance maxima at 492 and 342 nm, and is susceptible to photocatalytic isomerization and oxidation. This fluorophore was also detected in the eyecup extracts of Abca4(-/-)Rdh8(-/-) (Abca4 encodes ATP-binding cassette transporter 4 and Rdh8 encodes retinol dehydrogenase 8) mice, an AMD/recessive Stargardt's disease model. Excess amassing of pdA2E within RPE cells caused significant cell viability loss and membrane damage. The formation of pdA2E occurred when atRAL (all-trans-retinal) reacted with excess ethanolamine in the absence of acetic acid, and the process is likely to involve the participation of three atRAL molecules. Our findings suggest that endogenous pdA2E may serve as a sensitizer for yielding singlet oxygen and a singlet oxygen quencher, as well as a by-product of retinal metabolism, and its complete characterization facilitates the understanding of biosynthetic pathways by which adverse RPE lipofuscin constituents form.
Asunto(s)
Compuestos de Piridinio/metabolismo , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Retinoides/metabolismo , Animales , Bovinos , Humanos , Ratones , Ratones Noqueados , Compuestos de Piridinio/aislamiento & purificación , Retinaldehído/metabolismo , Retinoides/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Ferroptosis , Degeneración Retiniana , Animales , Ratones , Degeneración Retiniana/patología , Retinaldehído/metabolismo , Retinaldehído/farmacología , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ferroptosis/genética , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Transportadoras de Casetes de Unión a ATP/genéticaRESUMEN
Wandong cattle are an autochthonous Chinese breed used extensively for beef production. The breed tolerates extreme weather conditions and raw feed and is resistant to tick-borne diseases. However, the genetic basis of testis development and sperm production as well as breeding management is not well established in local cattle. Therefore, improving the reproductive efficiency of bulls via genetic selection is crucial as a single bull can breed thousands of cows through artificial insemination (AI). Testis development and spermatogenesis are regulated by hundreds of genes and transcriptomes. However, circular RNAs (circRNAs) are the key players in many biological developmental processes that have not been methodically described and compared between immature and mature stages in Bovine testes. In this study, we performed total RNA-seq and comprehensively analyzed the circRNA expression profiling of the testis samples of six bulls at 3 years and 3 months of developmental age. In total, 17,013 circRNAs were identified, of which 681 circRNAs (p-adjust < 0.05) were differentially expressed (DE). Among these DE circRNAs, 579 were upregulated and 103 were downregulated in calf and bull testes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the identified target genes were classified into three broad functional categories, including biological process, cellular component, and molecular function, and were enriched in the lysine degradation, cell cycle, and cell adhesion molecule pathways. The binding interactions between DE circRNAs and microRNAs (miRNAs) were subsequently constructed using bioinformatics approaches. The source genes ATM, CCNA1, GSK3B, KMT2C, KMT2E, NSD2, SUCLG2, QKI, HOMER1, and SNAP91 were found to be actively associated with bull sexual maturity and spermatogenesis. In addition, a real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed a strong correlation with the sequencing data. Moreover, the developed model of Bovine testes in the current study provides a suitable framework for understanding the mechanism of circRNAs in the development of testes and spermatogenesis.
RESUMEN
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.
Asunto(s)
Muerte Celular/genética , Regulación de la Expresión Génica , Inflamasomas/metabolismo , Degeneración Macular/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Epitelio Pigmentado de la Retina/ultraestructura , Western Blotting , Células Cultivadas , Humanos , Degeneración Macular/metabolismo , Degeneración Macular/patología , Microscopía Electrónica de Transmisión , Proteína con Dominio Pirina 3 de la Familia NLR/biosíntesis , ARN/genética , Epitelio Pigmentado de la Retina/metabolismo , Transducción de SeñalRESUMEN
Purpose: Nondegradable fluorophores that accumulate as deleterious lipofuscin of RPE are involved in pathological mechanisms leading to the degeneration of RPE in AMD. A2E, a major component of RPE lipofuscin, could cause damage to RPE cells. Nevertheless, all-trans-retinal dimer (atRAL dimer) was found to be much more abundant than that of A2E in eyes of Abca4-/-Rdh8-/- double-knockout (DKO) mice, a rodent model showing the typical characteristics of retinopathies in AMD patients. Our aim was to elucidate the effect and mechanism of atRAL dimer-induced RPE degeneration. Methods: Eyes harvested from C57BL/6J wild-type (WT) and Abca4-/-Rdh8-/- DKO mice were examined by HPLC. Cellular uptake, subcellular localization, 5-bromo-2-deoxyuridine (BrdU), Cdc25C, DNA strand breaks, mitochondrial membrane potential (ΔΨm), and cytochrome c were analyzed by fluorescence microscopy. Cellular toxicity was assayed by lactate dehydrogenase (LDH) assay and dead cell staining. Apoptosis and cell-cycle stages were detected by flow cytometry. Furthermore, in vitro and in vivo expression of proteins associated with cell cycle and apoptosis was measured by immunoblot assays. Results: All-trans-retinal dimer clearly could damage RPE cell membrane and inhibit the proliferation of RPE cells as well as induce DNA damage and cell-cycle arrest at the G2/M phase via activating the ATM/ATR-Chk2-p53 signaling pathway. Moreover, this di-retinal adduct triggered mitochondrion-associated apoptosis in RPE cells. Evidence from the cell-based experiments was also corroborated by a remarkable abnormality in expression of proteins associated with cell cycle (Cyclin B1 and Cdc2) and apoptosis (p53, Bcl-2 and Bax) in the RPE of Abca4-/-Rdh8-/- DKO mice. Conclusions: These findings suggest that atRAL dimer that accumulates beyond a critical level, facilitates age-dependent RPE degeneration.
Asunto(s)
Degeneración Retiniana/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Retinaldehído/análogos & derivados , Transportadoras de Casetes de Unión a ATP/genética , Animales , Apoptosis , Proliferación Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Citometría de Flujo , Humanos , Immunoblotting , Lipofuscina/metabolismo , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Retina/metabolismo , Retina/patología , Degeneración Retiniana/etiología , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/patología , Retinaldehído/metabolismoRESUMEN
The retinoid (visual) cycle is a complex enzymatic pathway that operates in the retina for the regeneration of 11-cis-retinal (11-cis-Ral), the inherent visual chromophore indispensable for vision. Deficiencies in the retinoid metabolism are involved in pathologic mechanisms of several forms of retinal diseases including age-related macular degeneration, Stargardt's disease, and Leber's congenital amaurosis, for which no effective cures presently exist. Nevertheless, the interference of abnormal retinoid metabolism with chemicals has been considered to be a promising strategy aimed at alleviating these retinal dysfunctions. Moreover, since gene therapy is gaining increasing importance in clinical practice, the modulation of key enzymes implicated with the retinoid cycle at a genetic level will hold great promise for the treatment of patients with degenerative diseases of the retina.
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Terapia Genética/métodos , Retina/metabolismo , Enfermedades de la Retina , Retinoides/metabolismo , Animales , Humanos , Enfermedades de la Retina/inducido químicamente , Enfermedades de la Retina/etiología , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/terapia , Retinoides/efectos adversos , Retinoides/farmacocinéticaRESUMEN
Effective clearance of all-trans-retinal (atRAL) from retinal pigment epithelial (RPE) cells is important for avoiding its cytotoxicity. However, the metabolism of atRAL in RPE cells is poorly clarified. The present study was designed to analyze metabolic products of atRAL and to compare the cytotoxicity of atRAL versus its derivative all-trans-retinal dimer (atRAL-dimer) in human RPE cells. We found that all-trans-retinol (atROL) and a mixture of atRAL condensation metabolites including atRAL-dimer and A2E were generated after incubating RPE cells with atRAL for 6h, and the amount of atRAL-dimer was significantly higher than that of A2E. In the eyes of Rdh8-/- Abca4-/- mice, a mouse model with defects in retinoid cycle that displays some symbolic characteristics of age-related macular degeneration (AMD), the level of atRAL-dimer was increased compared to wild-type mice, and was even much greater than that of A2E & isomers. The cytotoxicity of atRAL-dimer was reduced compared with its precursor atRAL. The latter could provoke intracellular reactive oxygen species (ROS) overproduction, increase the mRNA expression of several oxidative stress related genes (Nrf2, HO-1, and γ-GCSh), and induce ΔΨm loss in RPE cells. By contrast, the abilities of atRAL-dimer to induce intracellular ROS and oxidative stress were much weaker versus that of concentration-matched atRAL, and atRAL-dimer exhibited no toxic effect on mitochondrial function at higher concentrations. In conclusion, the formation of atRAL-dimer during atRAL metabolic process ameliorates the cytotoxicity of atRAL by reducing oxidative stress.
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Supervivencia Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Retinaldehído/análogos & derivados , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/genética , Oxidorreductasas de Alcohol/biosíntesis , Oxidorreductasas de Alcohol/genética , Animales , Línea Celular , ADN Complementario/biosíntesis , ADN Complementario/genética , Humanos , Degeneración Macular/genética , Degeneración Macular/prevención & control , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/citología , Retinaldehído/química , Retinaldehído/toxicidadRESUMEN
Excess accumulation of endogenous all-trans-retinal (atRAL) contributes to degeneration of the retinal pigment epithelium (RPE) and photoreceptor cells, and plays a role in the etiologies of age-related macular degeneration (AMD) and Stargardt's disease. In this study, we reveal that human RPE cells tolerate exposure of up to 5 µM atRAL without deleterious effects, but higher concentrations are detrimental and induce cell apoptosis. atRAL treatment significantly increased production of intracellular reactive oxygen species (ROS) and up-regulated mRNA expression of Nrf2, HO-1, and γ-GCSh within RPE cells, thereby causing oxidative stress. ROS localized to mitochondria and endoplasmic reticulum (ER). ER-resident molecular chaperone BiP, a marker of ER stress, was up-regulated at the translational level, and meanwhile, the PERK-eIF2α-ATF4 signaling pathway was activated. Expression levels of ATF4, CHOP, and GADD34 in RPE cells increased in a concentration-dependent manner after incubation with atRAL. Salubrinal, a selective inhibitor of ER stress, alleviated atRAL-induced cell death. The antioxidant N-acetylcysteine (NAC) effectively blocked RPE cell loss and ER stress activation, suggesting that atRAL-induced ROS generation is responsible for RPE degeneration and is an early trigger of ER stress. Furthermore, the mitochondrial transmembrane potential was lost after atRAL exposure, and was followed by caspase-3 activation and poly (ADP-ribose) polymerase cleavage. The results demonstrate that atRAL-driven ROS overproduction-induced ER stress is involved in cellular mitochondrial dysfunction and apoptosis of RPE cells.
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Estrés del Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Tretinoina/toxicidad , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular , Citoprotección , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Estrés del Retículo Endoplásmico/genética , Regulación de la Expresión Génica , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: Retinal-derived fluorophores that accumulate as RPE lipofuscin are implicated in pathological mechanisms of AMD. One component of RPE lipofuscin has been characterized as pdA2E, a pyridinium adduct derived from all-trans-retinal and excess ethanolamine. One-step preparation and biosynthetic studies of pdA2E and its novel isomer called isopdA2E are reported. METHODS: Biosynthetic reaction mixtures, RPE/choroids and neural retinas dissected from bovines, eyes harvested from Abca4(-/-)Rdh8(-/-) mice, irradiated samples, and enzyme-treated solutions were analyzed by HPLC, mass spectrometry, nuclear magnetic resonance spectroscopy, fluorescence spectrophotometry, and density functional theory (DFT). RESULTS: Optimization of the in vitro synthesis of pdA2E resulted in a biomimetic preparation of this pigment in a yield of 15%; this protocol also allowed the identification of isopdA2E, a double-bond isomer of pdA2E at the C13C14 position in bovine RPE lipofuscin. Interconversion between these two molecules occurs when either pdA2E or isopdA2E is exposed to light. A phospholipase D-based assay demonstrated the possibility of pdA2-PE being formed in neural retina and served as a precursor of pdA2E in the biosynthetic pathway. DFT calculations revealed that the 492-nm absorbance was assigned to the long arm of pdA2E/isopdA2E and the 340/342-nm absorbance to the short arm. Fluorescence efficiency of pdA2E and isopdA2E is very similar, but is much weaker in comparison with A2E, isoA2E, and iisoA2E. CONCLUSIONS: Our results facilitate the understanding of compositions and biosynthetic pathways of adverse RPE lipofuscin.
Asunto(s)
Coroides/metabolismo , Lipofuscina/química , Degeneración Retiniana/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Retinoides/biosíntesis , Animales , Biomimética , Bovinos , Modelos Animales de Enfermedad , Angiografía con Fluoresceína , Isomerismo , Ratones , Ratones Noqueados , Compuestos de Piridinio/química , Retinoides/químicaRESUMEN
Gene and drug therapies are being developed to alleviate vision loss in patients with Stargardt's disease and age-related macular degeneration (AMD). To evaluate the therapeutic effects of these treatments, organic solvents are routinely used to extract and quantify bisretinoid lipofuscin constituents, such as N-retinylidene-N-retinyl-ethanolamine (A2E) and all-trans-retinal dimer (ATR-dimer). By high-performance liquid chromatography (HPLC), we found that A2E and ATR-dimer were both altered by tetrahydrofuran (THF) and chloroform, but were stable in dimethyl sulfoxide (DMSO) or methanol (MeOH). In addition, cyclohexane and ethanol (EtOH) did not alter ATR-dimer, whereas an alteration of A2E occurred in EtOH. On the basis of these findings, we designed processes II-IV, generated by modifications of process I, a routine method to measure bisretinoid compounds in vivo. Extra amounts of either ATR-dimer or A2E in mouse eyecups were released by processes II-IV versus process I. Efforts to clarify the effects of organic solvents on lipofuscin pigments are important because such studies can guide the handling of these fluorophores in related experiments.