RESUMEN
Retinal pigment epithelium (RPE) cells have to phagocytose shed photoreceptor outer segments (POS) on a daily basis over the lifetime of an organism, but the mechanisms involved in the digestion and recycling of POS lipids are poorly understood. Although it was frequently assumed that peroxisomes may play an essential role, this was never investigated. Here, we show that global as well as RPE-selective loss of peroxisomal ß-oxidation in multifunctional protein 2 (MFP2) knockout mice impairs the digestive function of lysosomes in the RPE at a very early age, followed by RPE degeneration. This was accompanied by prolonged mammalian target of rapamycin activation, lipid deregulation, and mitochondrial structural anomalies without, however, causing oxidative stress or energy shortage. The RPE degeneration caused secondary photoreceptor death. Notably, the deterioration of the RPE did not occur in an Mfp2/rd1 mutant mouse line, characterized by absent POS shedding. Our findings prove that peroxisomal ß-oxidation in the RPE is essential for handling the polyunsaturated fatty acids present in ingested POS and shed light on retinopathy in patients with peroxisomal disorders. Our data also have implications for gene therapy development as they highlight the importance of targeting the RPE in addition to the photoreceptor cells.
Asunto(s)
Lisosomas , Epitelio Pigmentado de la Retina , Ratones , Humanos , Animales , Epitelio Pigmentado de la Retina/metabolismo , Lisosomas/metabolismo , Fagocitosis/genética , Estrés Oxidativo , Ratones Noqueados , MamíferosRESUMEN
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.
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Oftalmopatías , Neutrófilos , Humanos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Oftalmopatías/inmunología , Oftalmopatías/terapia , Animales , Trampas Extracelulares/metabolismo , Trampas Extracelulares/inmunología , Degeneración Macular/inmunología , Degeneración Macular/patología , Degeneración Macular/metabolismoRESUMEN
Age-related macular degeneration (AMD) is a disease that affects the macula - the central part of the retina. It is a leading cause of irreversible vision loss in the elderly. AMD onset is marked by the presence of lipid- and protein-rich extracellular deposits beneath the retinal pigment epithelium (RPE), a monolayer of polarized, pigmented epithelial cells located between the photoreceptors and the choroidal blood supply. Progression of AMD to the late nonexudative "dry" stage of AMD, also called geographic atrophy, is linked to progressive loss of areas of the RPE, photoreceptors, and underlying choriocapillaris leading to a severe decline in patients' vision. Differential susceptibility of macular RPE in AMD and the lack of an anatomical macula in most lab animal models has promoted the use of in vitro models of the RPE. In addition, the need for high throughput platforms to test potential therapies has driven the creation and characterization of in vitro model systems that recapitulate morphologic and functional abnormalities associated with human AMD. These models range from spontaneously formed cell line ARPE19, immortalized cell lines such as hTERT-RPE1, RPE-J, and D407, to primary human (fetal or adult) or animal (mouse and pig) RPE cells, and embryonic and induced pluripotent stem cell (iPSC) derived RPE. Hallmark RPE phenotypes, such as cobblestone morphology, pigmentation, and polarization, vary significantly betweendifferent models and culture conditions used in different labs, which would directly impact their usability for investigating different aspects of AMD biology. Here the AMD Disease Models task group of the Ryan Initiative for Macular Research (RIMR) provides a summary of several currently used in vitro RPE models, historical aspects of their development, RPE phenotypes that are attainable in these models, their ability to model different aspects of AMD pathophysiology, and pros/cons for their use in the RPE and AMD fields. In addition, due to the burgeoning use of iPSC derived RPE cells, the critical need for developing standards for differentiating and rigorously characterizing RPE cell appearance, morphology, and function are discussed.
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Atrofia Geográfica , Células Madre Pluripotentes Inducidas , Degeneración Macular , Adulto , Anciano , Animales , Técnicas de Cultivo de Célula , Atrofia Geográfica/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Degeneración Macular/metabolismo , Ratones , Epitelio Pigmentado de la Retina/metabolismo , PorcinosRESUMEN
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.
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Lipocalina 2/genética , Lipocalina 2/metabolismo , Degeneración Macular/genética , Degeneración Macular/patología , Trastornos de la Visión/genética , Animales , Modelos Animales de Enfermedad , Humanos , Inflamación/patología , Ratones , Retina/patología , Epitelio Pigmentado de la Retina/patología , Transducción de Señal , Trastornos de la Visión/patologíaRESUMEN
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.
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Adenoviridae/genética , Vectores Genéticos/administración & dosificación , Degeneración Macular/patología , Técnicas de Cultivo de Órganos/métodos , Epitelio Pigmentado de la Retina/citología , Transgenes , Animales , Células Cultivadas , Degeneración Macular/genética , Degeneración Macular/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Epitelio Pigmentado de la Retina/metabolismo , Transducción GenéticaRESUMEN
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.
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Ritmo Circadiano , Degeneración Macular/patología , Melatonina/biosíntesis , Epitelio Pigmentado de la Retina/patología , Animales , Humanos , Fagocitosis , Especies Reactivas de OxígenoRESUMEN
Cyberbullying is a newer phenomenon which is becoming more prevalent among adolescent students with the use of information technology. The objective of the study was to determine the prevalence of being cyberbullied among late adolescent and to study the behavior and the attitude on cyberbullying. It was a cross-sectional study conducted on 254 school students (15-19 years). Data were collected by self-administered questionnaire, and results were analyzed using SPSS version 20. About 210 (82.7%) students were using any form of social networking site and out of which 22 (10.5%) students were cyberbullied. Among those who were cyberbullied, the majority (16 [72.7%]) had no opinion and more than half (15 [68.2%]) sought their friends' help. Cyberbullying is emerging as a newer social problem in our country, where students' lack of awareness and understanding of it results in underreporting of cyberbullying incidents.
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Ciberacoso/estadística & datos numéricos , Redes Sociales en Línea , Adolescente , Conducta del Adolescente , Estudios Transversales , Femenino , Humanos , India , Masculino , Prevalencia , Adulto JovenRESUMEN
miRs play critical roles in oxidative stress-related retinopathy pathogenesis. miR-365 was identified in a previously constructed library from glyoxal-treated rat Müller cell. This report explores epigenetic alterations in Müller cells under oxidative stress to develop a novel therapeutic strategy. To examine the miR-365 expression pattern, in situ hybridization and quantitative RT-PCR were performed. Bioinformatical analysis and dual luciferase report assay were applied to identify and confirm target genes. Streptozotocin (STZ)-treated rats were used as the diabetic retinopathy (DR) model. Lentivirus-mediated anti-miR-365 was delivered subretinally and intravitreally into the rats' eyes. The functional and structural changes were evaluated by electroretinogram (ERG), histologically, and through examination of expression levels of metallopeptidase inhibitor 3 (Timp3), glial fibrillary acidic protein (Gfap), recoverin (Rcvrn) and vascular endothelia growth factor A (Vegfa). Oxidative stress factors and pro-inflammatory cytokines were analyzed. miR-365 expression was confirmed in the glyoxal-treated rat Müller cell line (glyoxal-treated rMC-1). In the retina, miR-365 mainly localized in the inner nuclear layer (INL). The increased miR-365 participated in Müller cell gliosis through oxidative stress aggravation, as observed in glyoxal-treated rMC-1 and DR rats before 6 weeks. Timp3 was a target and negatively regulated by miR-365. When miR-365 was inhibited, Timp3 expression was upregulated, Müller cell gliosis was alleviated, and retinal oxidative stress was attenuated. Visual function was also partially rescued as detected by ERG. miR-365 was found to be highly expressed in the retina and the abnormality of miR-365/Timp3 pathway is closely related to the pathology, like Müller gliosis, and the visual injury in DR. The mechanism might be through oxidative stress, and miR-365/Timp3 could be a potential therapeutic target for treating DR.
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Diabetes Mellitus Experimental/fisiopatología , Retinopatía Diabética/fisiopatología , MicroARNs/fisiología , Estrés Oxidativo/fisiología , Retina/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Análisis de Varianza , Animales , Far-Western Blotting , Células Cultivadas , Electrorretinografía , Células Ependimogliales/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
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Lipocalina 2/genética , Lisosomas/inmunología , Degeneración Macular/genética , FN-kappa B/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal , Factores de Edad , Animales , Autofagia , Coroides/inmunología , Coroides/metabolismo , Modelos Animales de Enfermedad , Humanos , Inflamación , Lipocalina 2/metabolismo , Lisosomas/metabolismo , Degeneración Macular/inmunología , Degeneración Macular/patología , Ratones , FN-kappa B/metabolismo , Neutrófilos/inmunología , Fagocitosis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Retina/inmunología , Retina/lesiones , Retina/metabolismo , Epitelio Pigmentado de la Retina/inmunología , Epitelio Pigmentado de la Retina/metabolismo , Regulación hacia ArribaRESUMEN
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.
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Proteínas del Ojo/metabolismo , Vítreo Primario Hiperplásico Persistente/embriología , Vítreo Primario Hiperplásico Persistente/metabolismo , Vasos Retinianos/anomalías , Vasos Retinianos/metabolismo , Cadena A de beta-Cristalina/metabolismo , Animales , Enfermedad Crónica , Humanos , Modelos BiológicosRESUMEN
The impairment of autophagic and proteasomal cleansing together with changes in pigmentation has been documented in retinal pigment epithelial (RPE) cell degeneration. However, the function and co-operation of these mechanisms in melanosome-containing RPE cells is still unclear. We show that inhibition of proteasomal degradation with MG-132 or autophagy with bafilomycin A1 increased the accumulation of premelanosomes and autophagic structures in human embryonic stem cell (hESC)-derived RPE cells. Consequently, upregulation of the autophagy marker p62 (also known as sequestosome-1, SQSTM1) was confirmed in Western blot and perinuclear staining. Interestingly, cells treated with the adenosine monophosphatedependent protein kinase activator, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), decreased the proteasome inhibitor-induced accumulation of premelanosomes, increased the amount of autophagosomes and eradicated the protein expression of p62 and LC3 (microtubule-associated protein 1A/1B-light chain 3). These results revealed that autophagic machinery is functional in hESC-RPE cells and may regulate cellular pigmentation with proteasomes.
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Autofagia/efectos de los fármacos , Células Madre Embrionarias Humanas/citología , Leupeptinas/farmacología , Macrólidos/farmacología , Pigmentación/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Línea Celular , Humanos , Melanosomas/efectos de los fármacos , Melanosomas/metabolismo , Epitelio Pigmentado de la Retina/citologíaRESUMEN
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.
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Autofagia/fisiología , Lisosomas/fisiología , Epitelio Pigmentado de la Retina/metabolismo , Animales , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/fisiología , Biogénesis de Organelos , Serina-Treonina Quinasas TOR/fisiologíaRESUMEN
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.
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Cristalinas/metabolismo , Células Epiteliales/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Catepsina D/metabolismo , Cristalinas/genética , Concentración de Iones de Hidrógeno , Immunoblotting , Lisosomas/metabolismo , Ratones Noqueados , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Epitelio Pigmentado de la Retina/citología , Cadena A de beta-CristalinaRESUMEN
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.
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Empalme Alternativo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Secuencia Conservada , Evolución Molecular , Exones , Regulación del Desarrollo de la Expresión Génica , Sistemas de Lectura Abierta , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Alineación de Secuencia , Análisis de Secuencia de ADN , Análisis de Secuencia de ProteínaRESUMEN
The World Health Organisation classification and the treatment protocol for the odontogenic keratocyst (OKC), previously referred to as the keratocystic odontogenic tumour, were examined based on a study of the literature. Because not all OKCs have an identifiable protein patched homolog mutation, the idea of changing the management protocol for OKC in response to this shift in tumour category was met with scepticism and was not widely adopted. This study's objective was to outline a successful management plan for an odontogenic keratocyst in a patient who was 23 years old. The procedure for therapy involved marsupialisation, which was followed by enucleation, peripheral osteotomy, and the injection of 5 FFU. Following a 2-year observation period (clinical and radiological monitoring), it was found that bone regeneration was normal and there was no sign of a recurrence.
RESUMEN
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.
RESUMEN
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.
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Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Lisosomas , Degeneración Macular , Proteínas Proto-Oncogénicas c-akt , Epitelio Pigmentado de la Retina , Transducción de Señal , Sirtuinas , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sirtuinas/metabolismo , Sirtuinas/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Degeneración Macular/genética , Humanos , Ratones , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Lisosomas/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Modelos Animales de Enfermedad , Células Madre Pluripotentes Inducidas/metabolismo , MasculinoRESUMEN
Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.
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Autofagia , Ferroptosis , Ferroptosis/fisiología , Humanos , Autofagia/fisiología , Animales , ConsensoRESUMEN
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.
Asunto(s)
Cristalinas/genética , Mutación , Fagosomas/genética , Epitelio Pigmentado de la Retina/metabolismo , Animales , Cristalinas/metabolismo , Fagosomas/metabolismo , Fagosomas/ultraestructura , Ratas , Ratas Sprague-Dawley , Epitelio Pigmentado de la Retina/ultraestructuraRESUMEN
VEGF(165) b is an anti-angiogenic form of VEGF(165) produced by alternative splicing. The localization of pro-angiogenic VEGF(165) and anti-angiogenic VEGF(165) b was investigated during development of the vasculatures in fetal human eyes from 7 to 21 weeks gestation (WG). The fetal vasculature of vitreous, which includes tunica vasculosa lentis (TVL), had moderate VEGF(165) immunoreactivity at 7WG and very little VEGF(165) b. Both forms were elevated at 12WG. VEGF(165) then decreased around 17WG when the TVL regresses but VEGF(165) b remained elevated. In choroid, VEGF(165) was present in forming choriocapillaris (CC) and retinal pigment epithelium (RPE) at 7WG while VEGF165b was present in CC and mesenchymal precursors within the choroidal stroma. By 21WG, both forms were elevated in RPE and choroidal blood vessels but VEGF(165) b was apical and VEGF(165) basal in RPE. Diffuse VEGF(165) immunoreactivity was prominent in 12WG innermost retina where blood vessels will form while VEGF(165) b was present in most CXCR4(+) progenitors in the inner neuroblastic layer and migrating angioblasts in the putative nerve fiber layer. By 21WG, VEGF(165) was present in nerve fibers and VEGF(165) b in the inner Muller cell process. The localization of VEGF(165) b was distinctly different from VEGF(165) both spatially and temporally and it was often associated with nucleus in progenitors.