RESUMO
Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people, with limited treatment options available for most patients. AMD involves the death of retinal pigment epithelium (RPE) and photoreceptor cells, with mitochondria dysfunction being a critical early event. In the current study, we utilized our unique resource of human donor RPE graded for AMD presence and severity to investigate proteome-wide dysregulation involved in early AMD. Organelle-enriched fractions of RPE were isolated from donors with early AMD (n = 45) and healthy age-matched controls (n = 32) and were analyzed by UHR-IonStar, an integrated proteomics platform enabling reliable and in-depth proteomic quantification in large cohorts. A total of 5941 proteins were quantified with excellent analytical reproducibility, and with further informatics analysis, many biological functions and pathways were found to be significantly dysregulated in donor RPE samples with early AMD. Several of these directly pinpointed changes in mitochondrial functions, e.g., translation, ATP metabolic process, lipid homeostasis, and oxidative stress. These novel findings highlighted the value of our proteomics investigation by allowing a better understanding of the molecular mechanisms underlying early AMD onset and facilitating both treatment development and biomarker discovery.
Assuntos
Degeneração Macular , Epitélio Pigmentado da Retina , Humanos , Idoso , Epitélio Pigmentado da Retina/metabolismo , Proteômica , Reprodutibilidade dos Testes , Degeneração Macular/metabolismo , Estresse OxidativoRESUMO
Genome-wide association studies (GWAS) have identified 52 independent variants at 34 genetic loci that are associated with age-related macular degeneration (AMD), the most common cause of incurable vision loss in the elderly worldwide. However, causal genes at the majority of these loci remain unknown. In this study, we performed whole exome sequencing of 264 individuals from 63 multiplex families with AMD and analyzed the data for rare protein-altering variants in candidate target genes at AMD-associated loci. Rare coding variants were identified in the CFH, PUS7, RXFP2, PHF12 and TACC2 genes in three or more families. In addition, we detected rare coding variants in the C9, SPEF2 and BCAR1 genes, which were previously suggested as likely causative genes at respective AMD susceptibility loci. Identification of rare variants in the CFH and C9 genes in our study validated previous reports of rare variants in complement pathway genes in AMD. We then extended our exome-wide analysis and identified rare protein-altering variants in 13 genes outside the AMD-GWAS loci in three or more families. Two of these genes, SCN10A and KIR2DL4, are of interest because variants in these genes also showed association with AMD in case-control cohorts, albeit not at the level of genome-wide significance. Our study presents the first large-scale, exome-wide analysis of rare variants in AMD. Further independent replications and molecular investigation of candidate target genes, reported here, would assist in gaining novel insights into mechanisms underlying AMD pathogenesis.
Assuntos
Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Degeneração Macular/genética , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Receptores KIR2DL4/genética , Idoso , Idoso de 80 Anos ou mais , Exoma/genética , Humanos , Degeneração Macular/patologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Sequenciamento do ExomaRESUMO
The retinal pigment epithelium is a pigmented monolayer of cells that help maintain a healthy retina. Loss of this essential cell layer is implicated in a number of visual disorders, including age-related macular degeneration (AMD). Utilizing primary RPE cultures to investigate disease is an important step in understanding disease mechanisms. However, the use of primary RPE cultures presents a number of challenges, including the limited number of cells available and the presence of auto-fluorescent pigment that interferes with quantifying fluorescent probes. Additionally, primary RPE are difficult to transfect with exogenous nucleic acids traditionally used for fluorescent imaging. To overcome these challenges, we used an adeno-associated viral (AAV) vector to express a pH sensitive fluorescent protein, mKeima, fused to the mitochondrial targeting sequence of cytochrome oxidase subunit 8A (mKeima-mito). mKeima-mito allows for quantification of mitochondrial autophagy (mitophagy) in live-cell time-lapse imaging experiments. We also developed an image analysis pipeline to selectively quantify mKeima-mito while removing the signal of auto-fluorescent pigment from the dataset by utilizing information from the mKeima fluorescent channels. These techniques are demonstrated in primary RPE cultures expressing mKeima-mito treated with 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP), an uncoupler that depolarizes the mitochondrial membrane and leads to mitochondrial fragmentation and mitophagy. The techniques outlined provide a roadmap for investigating disease mechanisms or the effect of treatments utilizing fluorescent probes in an important cell culture model.
Assuntos
Corantes Fluorescentes , Mitofagia , Células Cultivadas , Células Epiteliais , Corantes Fluorescentes/metabolismo , Humanos , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/metabolismoRESUMO
Strong experimental evidence from studies in human donor retinas and animal models supports the idea that the retinal pathology associated with age-related macular degeneration (AMD) involves mitochondrial dysfunction and consequent altered retinal metabolism. This chapter provides a brief overview of mitochondrial structure and function, summarizes evidence for mitochondrial defects in AMD, and highlights the potential ramifications of these defects on retinal health and function. Discussion of mitochondrial haplogroups and their association with AMD brings to light how mitochondrial genetics can influence disease outcome. As one of the most metabolically active tissues in the human body, there is strong evidence that disruption in key metabolic pathways contributes to AMD pathology. The section on retinal metabolism reviews cell-specific metabolic differences and how the metabolic interdependence of each retinal cell type creates a unique ecosystem that is disrupted in the diseased retina. The final discussion includes strategies for therapeutic interventions that target key mitochondrial pathways as a treatment for AMD.
Assuntos
DNA Mitocondrial , Degeneração Macular , Animais , DNA Mitocondrial/metabolismo , Ecossistema , Humanos , Degeneração Macular/genética , Degeneração Macular/metabolismo , Mitocôndrias/genética , Retina , Epitélio Pigmentado da Retina/metabolismoRESUMO
Oxidative modification of proteins can perturb their structure and function, often compromising cellular viability. Such modifications include lipid-derived adducts (e.g., 4-hydroxynonenal (HNE) and carboxyethylpyrrole (CEP)) as well as nitrotyrosine (NTyr). We compared the retinal proteome and levels of such modifications in the AY9944-treated rat model of Smith-Lemli-Opitz syndrome (SLOS), in comparison to age-matched controls. Retinas harvested at 3 months of age were either subjected to proteomic analysis or to immuno-slot blot analysis, the latter probing blots with antibodies raised against HNE, CEP, and NTyr, followed by quantitative densitometry. HNE modification of retinal proteins was markedly (>9-fold) higher in AY9944-treated rats compared to controls, whereas CEP modification was only modestly (≤2-fold) greater, and NTyr modification was minimal and exhibited no difference as a function of AY9944 treatment. Anti-HNE immunoreactivity was greatest in the plexiform and ganglion cell layers, but also present in the RPE, choroid, and photoreceptor outer segment layer in AY9944-treated rats; control retinas showed minimal HNE labeling. 1D-PAGE/Western blot analysis of rod outer segment (ROS) membranes revealed HNE modification of both opsin and ß-transducin. Proteomic analysis revealed the differential expression of several retinal proteins as a consequence of AY9944 treatment. Upregulated proteins included those involved in chaperone/protein folding, oxidative and cellular stress responses, transcriptional regulation, and energy production. ßA3/A1 Crystallin, which has a role in regulation of lysosomal acidification, was down-regulated. Hence, oxidative modification of retinal proteins occurs in the SLOS rat model, in addition to the previously described oxidation of lipids. The results are discussed in the context of the histological and physiological changes that occur in the retina in the SLOS rat model.
Assuntos
Modelos Animais de Doenças , Opsinas/metabolismo , Estresse Oxidativo , Retina/metabolismo , Síndrome de Smith-Lemli-Opitz/metabolismo , Transducina/metabolismo , Aldeídos/metabolismo , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Feminino , Gravidez , Proteômica , Ratos , Ratos Sprague-Dawley , Dicloridrato de trans-1,4-Bis(2-clorobenzaminometil)ciclo-hexano/farmacologiaRESUMO
The blood retinal barrier (BRB) closely regulates the retinal microenvironment. Its compromise leads to the accumulation of retinal fluid containing potentially harmful plasma components. While eyes with non-exudative age-related macular degeneration (AMD) were previously felt to have an intact BRB, we propose that the BRB in non-exudative AMD eyes may be subclinically compromised, allowing entry of retina-toxic plasma proteins. We test this hypothesis by measuring retinal levels of abundant plasma proteins that should not cross the intact BRB. Two cohorts of frozen, post mortem neurosensory retinas were studied by Western analysis. One cohort from Alabama had 4 normal controls and 4 eyes with various forms of AMD. Another cohort from Minnesota had 5 intermediate AMD eyes and 5 normals. Both cohorts were age/post mortem interval (PMI) matched. The non-exudative AMD retinas in the Alabama cohort had significantly higher levels of albumin and complement component 9 (C9) than normal controls. The positive control exudative AMD donor retina had higher levels of all but one serum protein. In both macular and peripheral neurosensory retina samples, intermediate AMD retinas in the Minnesota cohort had significantly higher levels of albumin, fibrinogen, IgG, and C9 than controls. Our results suggest that there may be moderate subclinical BRB leakage in non-exudative AMD. Potentially harmful plasma components including complement or iron could enter the neurosensory retina in AMD patients prior to advanced disease. Thus, therapies aiming to stabilize the BRB might have a role in the management of non-exudative AMD.
Assuntos
Proteínas Sanguíneas/metabolismo , Atrofia Geográfica/sangue , Retina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Barreira Hematorretiniana/fisiologia , Western Blotting , Complemento C9/metabolismo , Exsudatos e Transudatos , Feminino , Fibrinogênio/metabolismo , Humanos , Imunoglobulina G/metabolismo , Masculino , Albumina Sérica/metabolismoRESUMO
Rhinovirus (RV) infection is involved in acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). RV primarily infects upper and lower airway epithelium. Immunoproteasomes (IP) are proteolytic machineries with multiple functions including the regulation of MHC class I antigen processing during viral infection. However, the role of IP in RV infection has not been explored. We sought to investigate the expression and function of IP during airway RV infection. Primary human tracheobronchial epithelial (HTBE) cells were cultured at air-liquid interface (ALI) and treated with RV16, RV1B, or interferon (IFN)-λ in the absence or presence of an IP inhibitor (ONX-0914). IP gene (i.e. LMP2) deficient mouse tracheal epithelial cells (mTECs) were cultured for the mechanistic studies. LMP2-deficient mouse model was used to define the in vivo role of IP in RV infection. IP subunits LMP2 and LMP7, antiviral genes MX1 and OAS1 and viral load were measured. Both RV16 and RV1B significantly increased the expression of LMP2 and LMP7 mRNA and proteins, and IFN-λ mRNA in HTBE cells. ONX-0914 down-regulated MX1 and OAS1, and increased RV16 load in HTBE cells. LMP2-deficient mTECs showed a significant increase in RV1B load compared with the wild-type (WT) cells. LMP2-deficient (compared with WT) mice increased viral load and neutrophils in bronchoalveolar lavage (BAL) fluid after 24 h of RV1B infection. Mechanistically, IFN-λ induction by RV infection contributed to LMP2 and LMP7 up-regulation in HTBE cells. Our data suggest that IP are induced during airway RV infection, which in turn may serve as an antiviral and anti-inflammatory mechanism.
Assuntos
Células Epiteliais/imunologia , Infecções por Picornaviridae/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Rhinovirus/imunologia , Animais , Asma/enzimologia , Asma/imunologia , Asma/virologia , Linhagem Celular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Células Epiteliais/enzimologia , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Camundongos Knockout , Oligopeptídeos/farmacologia , Infecções por Picornaviridae/enzimologia , Infecções por Picornaviridae/virologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Sistema Respiratório/enzimologia , Sistema Respiratório/imunologia , Sistema Respiratório/virologia , Rhinovirus/fisiologiaRESUMO
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.
Assuntos
Lipocalina-2/genética , Lisossomos/imunologia , Degeneração Macular/genética , NF-kappa B/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Fatores Etários , Animais , Autofagia , Corioide/imunologia , Corioide/metabolismo , Modelos Animais de Doenças , Humanos , Inflamação , Lipocalina-2/metabolismo , Lisossomos/metabolismo , Degeneração Macular/imunologia , Degeneração Macular/patologia , Camundongos , NF-kappa B/metabolismo , Neutrófilos/imunologia , Fagocitose , Proteínas Proto-Oncogênicas c-akt/metabolismo , Retina/imunologia , Retina/lesões , Retina/metabolismo , Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/metabolismo , Regulação para CimaRESUMO
Age-related macular degeneration (AMD) is the leading cause of blindness among older adults in the developed world. Although the pathological mechanisms have not been definitively elucidated, evidence suggests a key role for mitochondrial (mt) dysfunction. The current study used our unique collection of human retinal samples graded for the donor's stage of AMD to address fundamental questions about mtDNA damage in the retina. To evaluate the distribution of mtDNA damage in the diseased retina, damage in the retinal pigment epithelium (RPE) and neural retina from individual donors were compared. To directly test a long-held belief that the macula is selectively damaged with AMD, RPE mtDNA damage was measured in the macula and peripheral sections from individual donors. Small segments of the entire mt genome were examined to determine whether specific regions are preferentially damaged. Our results show that mtDNA damage is limited to the RPE, equivalent mtDNA damage is found in the macular and peripheral RPE, and sites of damage are localized to regions of the mt genome that may impact mt function. These results provide a scientific basis for targeting the RPE mitochondria with therapies that protect and enhance mt function as a strategy for combating AMD.
Assuntos
Envelhecimento/metabolismo , Sistemas de Liberação de Medicamentos , Marcação de Genes , Degeneração Macular/metabolismo , Mitocôndrias/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , DNA Mitocondrial/metabolismo , Sistemas de Liberação de Medicamentos/tendências , Feminino , Marcação de Genes/tendências , Humanos , Degeneração Macular/patologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia , Epitélio Pigmentado da Retina/patologiaRESUMO
Age-related macular degeneration (AMD) is a major cause of blindness among the elderly in the developed world. Genetic analysis of AMD has identified 34 high-risk loci associated with AMD. The genes at these high risk loci belong to diverse biological pathways, suggesting different mechanisms leading to AMD pathogenesis. Thus, therapies targeting a single pathway for all AMD patients will likely not be universally effective. Recent evidence suggests defects in mitochondria (mt) of the retinal pigment epithelium (RPE) may constitute a key pathogenic event in some AMD patients. The purpose of this study is to determine if individuals with a specific genetic background have a greater propensity for mtDNA damage. We used human eyebank tissues from 76 donors with AMD and 42 age-matched controls to determine the extent of mtDNA damage in the RPE that was harvested from the macula using a long extension polymerase chain reaction assay. Genotype analyses were performed for ten common AMD-associated nuclear risk alleles (ARMS2, TNFRSF10A, CFH, C2, C3, APOE, CETP, LIPC, VEGF and COL10A1) and mtDNA haplogroups. Sufficient samples were available for genotype association with mtDNA damage for TNFRSF10A, CFH, CETP, VEGFA, and COL10A1. Our results show that AMD donors carrying the high risk allele for CFH (C) had significantly more mtDNA damage compared with donors having the wild-type genetic profile. The data from an additional 39 donors (12 controls and 27 AMD) genotyped for CFH alleles further supported these findings. Taken together, these studies provide the rationale for a more personalized approach for treating AMD by uncovering a significant correlation between the CFH high risk allele and accelerated mtDNA damage. Patients harboring this genetic risk factor may benefit from therapies that stabilize and protect the mt in the RPE.
Assuntos
Fator H do Complemento/genética , Dano ao DNA/fisiologia , DNA Mitocondrial , Degeneração Macular/genética , Epitélio Pigmentado da Retina , Idoso , Idoso de 80 Anos ou mais , Alelos , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/fisiopatologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Immune system cells are known to affect loss of neurons due to injury or disease. Recruitment of immune cells following retinal/CNS injury has been shown to affect the health and survival of neurons in several models. We detected close, physical contact between dendritic cells and retinal ganglion cells following an optic nerve crush, and sought to understand the underlying mechanisms. METHODS: CD11c-DTR/GFP mice producing a chimeric protein of diphtheria toxin receptor (DTR) and GFP from a transgenic CD11c promoter were used in conjunction with mice deficient in MyD88 and/or TRIF. Retinal ganglion cell injury was induced by an optic nerve crush, and the resulting interactions of the GFPhi cells and retinal ganglion cells were examined. RESULTS: Recruitment of GFPhi dendritic cells to the retina was significantly compromised in MyD88 and TRIF knockout mice. GFPhi dendritic cells played a significant role in clearing fluorescent-labeled retinal ganglion cells post-injury in the CD11c-DTR/GFP mice. In the TRIF and MyD88 deficient mice, the resting level of GFPhi dendritic cells was lower, and their influx was reduced following the optic nerve crush injury. The reduction in GFPhi dendritic cell numbers led to their replacement in the uptake of fluorescent-labeled debris by GFPlo microglia/macrophages. Depletion of GFPhi dendritic cells by treatment with diphtheria toxin also led to their displacement by GFPlo microglia/macrophages, which then assumed close contact with the injured neurons. CONCLUSIONS: The contribution of recruited cells to the injury response was substantial, and regulated by MyD88 and TRIF. However, the presence of these adaptor proteins was not required for interaction with neurons, or the phagocytosis of debris. The data suggested a two-niche model in which resident microglia were maintained at a constant level post-optic nerve crush, while the injury-stimulated recruitment of dendritic cells and macrophages led to their transient appearance in numbers equivalent to or greater than the resident microglia.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/deficiência , Movimento Celular/genética , Células Dendríticas/fisiologia , Fator 88 de Diferenciação Mieloide/deficiência , Células Ganglionares da Retina/patologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Antígenos de Diferenciação/metabolismo , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Células Dendríticas/efeitos dos fármacos , Toxina Diftérica/farmacologia , Modelos Animais de Doenças , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Células Mieloides/fisiologia , Fator 88 de Diferenciação Mieloide/genética , Traumatismos do Nervo Óptico/patologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Fatores de Tempo , Vias Visuais/patologiaRESUMO
Increased proteasome activity has been implicated in the atrophy and deterioration associated with dystrophic muscles of Duchenne muscular dystrophy (DMD). While proteasome inhibitors show promise in the attenuation of muscle degeneration, proteasome inhibition-induced toxicity was a major drawback of this therapeutic strategy. Inhibitors that selectively target the proteasome subtype that is responsible for the loss in muscle mass and quality would reduce side effects and be less toxic. This study examined proteasome activity and subtype populations, along with muscle function, morphology and damage in wild-type (WT) mice and two murine models of DMD, dystrophin-deficient (MDX) and dystrophin- and utrophin-double-knockout (DKO) mice. We found that immunoproteasome content was increased in dystrophic muscles while the total proteasome content was unchanged among the three genotypes of mice. Proteasome proteolytic activity was elevated in dystrophic muscles, especially in DKO mice. These mice also exhibited more severe muscle atrophy than either WT or MDX mice. Muscle damage and regeneration, characterized by the activity of muscle creatine kinase in the blood and the percentage of central nuclei were equally increased in dystrophic mice. Accordingly, the overall muscle function was similarly reduced in both dystrophic mice compared with WT. These data demonstrated that there was transformation of standard proteasomes to immunoproteasomes in dystrophic muscles. In addition, DKO that showed greatest increase in proteasome activities also demonstrated more severe atrophy compared with MDX and WT. These results suggest a putative role for the immunoproteasome in muscle deterioration associated with DMD and provide a potential target for therapeutic intervention.
Assuntos
Imunoproteínas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/enzimologia , Músculo Esquelético/imunologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/enzimologia , Distrofia Muscular Animal/imunologia , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular de Duchenne/enzimologia , Distrofia Muscular de Duchenne/imunologia , Distrofia Muscular de Duchenne/fisiopatologiaRESUMO
DNA methylation provides a crucial epigenetic mark linking genetic variations to environmental influence. We have analyzed array-based DNA methylation profiles of 160 human retinas with co-measured RNA-seq and >8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 methylation quantitative trait loci and 12,505 expression quantitative trait loci) and 13,747 DNA methylation loci affecting gene expression, with over one-third specific to the retina. Methylation and expression quantitative trait loci show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration. Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of macular degeneration pathology by genotype-environment interaction in retina.
Assuntos
Metilação de DNA , Degeneração Macular , Humanos , Metilação de DNA/genética , Epigênese Genética , Epigenoma , Degeneração Macular/genética , RetinaRESUMO
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.
Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Lisossomos , Degeneração Macular , Proteínas Proto-Oncogênicas c-akt , Epitélio Pigmentado da Retina , Transdução de Sinais , Sirtuínas , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sirtuínas/metabolismo , Sirtuínas/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Degeneração Macular/genética , Humanos , Camundongos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Lisossomos/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Modelos Animais de Doenças , Células-Tronco Pluripotentes Induzidas/metabolismo , MasculinoRESUMO
Macroautophagy/autophagy is a key process in the maintenance of cellular homeostasis. The age-dependent decline in retinal autophagy has been associated with photoreceptor degeneration. Retinal dysfunction can also result from damage to the retinal pigment epithelium (RPE), as the RPE-retina constitutes an important metabolic ecosystem that must be finely tuned to preserve visual function. While studies of mice lacking essential autophagy genes have revealed a predisposition to retinal degeneration, the consequences of a moderate reduction in autophagy, similar to that which occurs during physiological aging, remain unclear. Here, we described a retinal phenotype consistent with accelerated aging in mice carrying a haploinsufficiency for Ambra1, a pro-autophagic gene. These mice showed protein aggregation in the retina and RPE, metabolic underperformance, and premature vision loss. Moreover, Ambra1+/gt mice were more prone to retinal degeneration after RPE stress. These findings indicate that autophagy provides crucial support to RPE-retinal metabolism and protects the retina against stress and physiological aging.Abbreviations : 4-HNE: 4-hydroxynonenal; AMBRA1: autophagy and beclin 1 regulator 1, AMD: age-related macular degeneration;; GCL: ganglion cell layer; GFAP: glial fibrillary acidic protein; GLUL: glutamine synthetase/glutamate-ammonia ligase; HCL: hierarchical clustering; INL: inner nuclear layer; IPL: inner plexiform layer; LC/GC-MS: liquid chromatography/gas chromatography-mass spectrometry; MA: middle-aged; MTDR: MitoTracker Deep Red; MFI: mean fluorescence intensity; NL: NH4Cl and leupeptin; Nqo: NAD(P)H quinone dehydrogenase; ONL: outer nuclear layer; OPL: outer plexiform layer; OP: oscillatory potentials; OXPHOS: oxidative phosphorylation; PCR: polymerase chain reaction; PRKC/PKCα: protein kinase C; POS: photoreceptor outer segment; RGC: retinal ganglion cells; RPE: retinal pigment epithelium; SI: sodium iodate; TCA: tricarboxylic acid.
Assuntos
Degeneração Retiniana , Camundongos , Animais , Degeneração Retiniana/genética , Ecossistema , Haploinsuficiência , Autofagia/genética , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Age-related macular degeneration (AMD), the leading cause of blindness in elderly populations, involves the loss of central vision due to progressive dysfunction of the retinal pigment epithelium (RPE) and subsequent loss of light-sensing photoreceptors. While age is a key risk factor, not every aged individual develops AMD. Thus, the critical question is what specific cellular changes tip the balance from healthy aging to disease. To distinguish between changes associated with aging and AMD, we compared the RPE proteome in human eye bank tissue from nondiseased donors during aging (n = 50, 29-91 years) and in donors with AMD (n = 36) compared to age-matched donors without disease (n = 28). Proteins from RPE cells were separated on two-dimensional gels, analyzed for content, and identified using mass spectrometry. A total of 58 proteins displayed significantly altered content with either aging or AMD. Proteins involved in metabolism, protein turnover, stress response, and cell death were altered with both aging and AMD. However, the direction of change was predominantly opposite. With aging, we detected an overall decrease in metabolism and reductions in stress-associated proteins, proteases, and chaperones. With AMD, we observed upregulation of metabolic proteins involved in glycolysis, TCA, and fatty acid metabolism, with a concurrent decline in oxidative phosphorylation, suggesting a reprogramming of energy utilization. Additionally, we detected upregulation of proteins involved in the stress response and protein turnover. Predicted upstream regulators also showed divergent results, with inhibition of inflammation and immune response with aging and activation of these processes with AMD. Our results support the idea that AMD is not simply advanced aging but rather the culmination of perturbed protein homeostasis, defective bioenergetics, and increased oxidative stress within the aging RPE, exacerbated by environmental factors and the genetic background of an individual.
Assuntos
Degeneração Macular , Epitélio Pigmentado da Retina , Idoso , Humanos , Epitélio Pigmentado da Retina/metabolismo , Degeneração Macular/metabolismo , Envelhecimento , Estresse Oxidativo , Fosforilação OxidativaRESUMO
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in developed countries, characterized by the death of retinal pigment epithelial (RPE) cells and photoreceptors. Previous studies report an accumulation of damaged and dysfunctional mitochondria in RPE of human donors with AMD. Understanding how damaged mitochondria accumulate in AMD is an important step in discovering disease mechanisms and identifying therapeutic targets. In this report, we assessed mitochondrial fission and fusion by quantifying proteins and measured mitochondrial autophagy (mitophagy) via protein analysis and advanced imaging techniques using mitochondrial targeted mKeima in primary human RPE from donors with or without AMD. We report disease-specific differences in mitochondrial proteins that regulate fission, fusion, and mitophagy that were present at baseline and with treatments to stimulate these pathways. Data suggest AMD RPE utilize receptor-mediated mitophagy as a compensatory mechanism for deficits in the ubiquitin-mediated mitophagy pathway. These changes in mitochondrial homeostasis could lead to the buildup of damaged and dysfunctional mitochondria observed in the RPE of AMD donors.
Assuntos
Degeneração Macular , Epitélio Pigmentado da Retina , Humanos , Epitélio Pigmentado da Retina/metabolismo , Dinâmica Mitocondrial , Estresse Oxidativo , Degeneração Macular/metabolismo , AutofagiaRESUMO
Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. No universally effective treatments exist for atrophic or "dry" AMD, which results from loss of the retinal pigment epithelium (RPE) and photoreceptors and accounts for ≈80% of all AMD patients. Prior studies provide evidence for the involvement of mitochondrial dysfunction in AMD pathology. This study used induced pluripotent stem cell (iPSC) RPE derived from five AMD patients to test the efficacy of three drugs (AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), Metformin, trehalose) that target key processes in maintaining optimal mitochondrial function. The patient iPSC-RPE lines were used in a proof-of-concept drug screen, utilizing an analysis of RPE mitochondrial function following acute and extended drug exposure. Results show considerable variability in drug response across patient cell lines, supporting the need for a personalized medicine approach for treating AMD. Furthermore, our results demonstrate the feasibility of using iPSC-RPE from AMD patients to develop a personalized drug treatment regime and provide a roadmap for the future clinical management of AMD.