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Although dysregulated cholesterol metabolism predisposes aging tissues to inflammation and a plethora of diseases, the underlying molecular mechanism remains poorly defined. Here, we show that metabolic and genotoxic stresses, convergently acting through liver X nuclear receptor, upregulate CD38 to promote lysosomal cholesterol efflux, leading to nicotinamide adenine dinucleotide (NAD+) depletion in macrophages. Cholesterol-mediated NAD+ depletion induces macrophage senescence, promoting key features of age-related macular degeneration (AMD), including subretinal lipid deposition and neurodegeneration. NAD+ augmentation reverses cellular senescence and macrophage dysfunction, preventing the development of AMD phenotype. Genetic and pharmacological senolysis protect against the development of AMD and neurodegeneration. Subretinal administration of healthy macrophages promotes the clearance of senescent macrophages, reversing the AMD disease burden. Thus, NAD+ deficit induced by excess intracellular cholesterol is the converging mechanism of macrophage senescence and a causal process underlying age-related neurodegeneration.
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ADP-Ribosil Ciclase 1 , Senescência Celular , Colesterol , Receptores X do Fígado , Macrófagos , Camundongos Endogâmicos C57BL , NAD , NAD/metabolismo , Animais , Receptores X do Fígado/metabolismo , Macrófagos/metabolismo , Senescência Celular/efeitos dos fármacos , Colesterol/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , ADP-Ribosil Ciclase 1/genética , Camundongos , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Lisossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , MasculinoRESUMO
Purpose: Choroidal neovascularization (CNV) accounts for the majority of severe vision loss in neovascular age-related macular degeneration (AMD). Despite therapies that target VEGF, patients are often under-responsive, require frequent eye injections to control disease, and eventually lose some vision despite chronic therapy implicating a multifactorial etiology in treatment response. Genetic studies implicate systemic immunity in AMD and systemic immune cells accumulate within CNV lesions, yet a role for these cells in anti-VEGF response remains undetermined. The purpose of this study was to identify transcriptional signatures of circulating immune cells that are associated with high anti-VEGF treatment burden. Design: Experimental pilot study. Participants: Patients with neovascular AMD seen at Washington University School of Medicine in St. Louis and BJC Health System. Methods: We profiled by single cell RNA sequencing the peripheral blood mononuclear cells of 27 treatment-experienced patients with wet AMD. We stratified this cohort into 2 groups with low and high treatment burden (≤ 5 or ≥ 6 injections in the past 12 months, respectively). Main Outcome Measures: Identification of immune cells associated with high treatment burden. Results: Gene expression signature of CD16+ monocytes may be associated with high treatment burden. Conclusions: These studies delineate potential signatures of circulating immune cells that may be associated with high treatment burden in neovascular AMD, potentially informing the development of diagnostic predictors of anti-VEGF response and new precision medicine-based approaches to complement anti-VEGF therapies. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of α, ß, and γ subunits. The independent functions of the 2 isoforms of the catalytic α subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons, such as photoreceptors. Here, we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, showed altered levels of cGMP, GTP, and ATP, suggesting isoform-specific regulation of photoreceptor metabolism. Furthermore, PRKAA2-deficient mice displayed visual functional deficits on electroretinography and photoreceptor outer segment structural abnormalities on transmission electron microscopy consistent with neuronal dysfunction, but not neurodegeneration. Phosphoproteomics identified inosine monophosphate dehydrogenase (IMPDH) as a molecular driver of PRKAA2-specific photoreceptor dysfunction, and inhibition of IMPDH improved visual function in Prkaa2 rod photoreceptor-knockout mice. These findings highlight a therapeutically targetable PRKAA2 isoform-specific function of AMPK in regulating photoreceptor metabolism and function through a potentially previously uncharacterized mechanism affecting IMPDH activity.
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Proteínas Quinases Ativadas por AMP , Células Fotorreceptoras Retinianas Bastonetes , Animais , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Isoformas de Proteínas/metabolismo , Eletrorretinografia , Camundongos Knockout , Trifosfato de Adenosina/metabolismoRESUMO
Age-related macular degeneration (AMD) is a leading cause of blindness featuring pathogenic neovascularization of the choroidal vasculature (CNV). Although systemic immunity plays a role in AMD, the ocular signals that recruit and activate immune cells remain poorly defined. Using single-cell RNA sequencing, we prospectively profile peripheral blood mononuclear cells from 65 individuals including AMD and controls, which we integrate with existing choroid data. We generate a network of choroid-peripheral immune interactions dysregulated in AMD, including known AMD-relevant gene vascular endothelial growth factor (VEGF) receptor 2. Additionally, we find CYR61 is upregulated in choroidal veins and may signal to circulating monocytes. In mice, we validate that CYR61 is abundant in endothelial cells within CNV lesions neighboring monocyte-derived macrophages. Mechanistically, CYR61 activates macrophage anti-angiogenic gene expression, and ocular Cyr61 knockdown increases murine CNV size, indicating CYR61 inhibits CNV. This study highlights the potential of multi-tissue human datasets to identify disease-relevant and potentially therapeutically modifiable targets.
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Neovascularização de Coroide , Degeneração Macular , Humanos , Camundongos , Animais , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Leucócitos Mononucleares/metabolismo , Células Endoteliais/metabolismo , Degeneração Macular/genética , Degeneração Macular/complicações , Degeneração Macular/metabolismo , Corioide/metabolismo , Corioide/patologiaRESUMO
Diabetic retinopathy (DR) is a neurovascular complication of diabetes. Recent investigations have suggested that early degeneration of the neuroretina may occur prior to the appearance of microvascular changes; however, the mechanisms underlying this neurodegeneration have been elusive. Microglia are the predominant resident immune cell in the retina and adopt dynamic roles in disease. Here, we show that ablation of retinal microglia ameliorates visual dysfunction and neurodegeneration in a type I diabetes mouse model. We also provide evidence of enhanced microglial contact and engulfment of amacrine cells, ultrastructural modifications, and transcriptome changes that drive inflammation and phagocytosis. We show that CD200-CD200R signaling between amacrine cells and microglia is dysregulated during early DR and that targeting CD200R can attenuate high glucose-induced inflammation and phagocytosis in cultured microglia. Last, we demonstrate that targeting CD200R in vivo can prevent visual dysfunction, microglia activation, and retinal inflammation in the diabetic mouse. These studies provide a molecular framework for the pivotal role that microglia play in early DR pathogenesis and identify a potential immunotherapeutic target for treating DR in patients.
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Diabetes Mellitus , Retinopatia Diabética , Animais , Humanos , Camundongos , Diabetes Mellitus/metabolismo , Retinopatia Diabética/metabolismo , Inflamação/metabolismo , Microglia/metabolismo , Retina/metabolismo , Transdução de SinaisRESUMO
Macrophages are innate immune cells that patrol tissues and are the first responders to detect infection. They orchestrate the host immune response in eliminating invading pathogens and the subsequent transition from inflammation to tissue repair. Macrophage dysfunction contributes to age-related pathologies, including low-grade inflammation in advanced age that is termed "inflammaging." Our laboratory has previously identified that macrophage expression of a fatty acid desaturase, stearoyl-CoA desaturase 2 (SCD2), declines with age. Herein, we delineate the precise cellular effects of SCD2 deficiency in murine macrophages. We found that deletion of Scd2 from macrophages dysregulated basal and bacterial lipopolysaccharide (LPS)-stimulated transcription of numerous inflammation-associated genes. Specifically, deletion of Scd2 from macrophages decreased basal and LPS-induced expression of Il1b transcript that corresponded to decreased production of precursor IL1B protein and release of mature IL1B. Furthermore, we identified disruptions in autophagy and depletion of unsaturated cardiolipins in SCD2-deficient macrophages. To assess the functional relevance of SCD2 in the macrophage response to infection, we challenged SCD2-deficient macrophages with uropathogenic Escherichia coli and found that there was impaired clearance of intracellular bacteria. This increased burden of intracellular bacteria was accompanied by increased release of pro-inflammatory cytokines IL6 and TNF but decreased IL1B. Taken together, these results indicate that macrophage expression of Scd2 is necessary for maintaining the macrophage response to inflammatory stimuli. This link between fatty acid metabolism and fundamental macrophage effector functions may potentially be relevant to diverse age-related pathologies. IMPORTANCE Macrophages are immune cells that respond to infection, but their dysfunction is implicated in many age-related diseases. Recent evidence showed that macrophage expression of a fatty acid enzyme, stearoyl-CoA desaturase 2, declines in aged organisms. In this work, we characterize the effects when stearoyl-CoA desaturase 2 is deficient in macrophages. We identify aspects of the macrophage inflammatory response to infection that may be affected when expression of a key fatty acid enzyme is decreased, and these findings may provide cellular insight into how macrophages contribute to age-related diseases.
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Lipopolissacarídeos , Estearoil-CoA Dessaturase , Animais , Camundongos , Sequência de Bases , Ácidos Graxos/metabolismo , Inflamação/genética , Macrófagos/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismoRESUMO
Many epithelial compartments undergo constitutive renewal in homeostasis but activate unique regenerative responses following injury. The clear corneal epithelium is crucial for vision and is renewed from limbal stem cells (LSCs). Using single-cell RNA sequencing, we profiled the mouse corneal epithelium in homeostasis, aging, diabetes, and dry eye disease (DED), where tear deficiency predisposes the cornea to recurrent injury. In homeostasis, we capture the transcriptional states that accomplish continuous tissue turnover. We leverage our dataset to identify candidate genes and gene networks that characterize key stages across homeostatic renewal, including markers for LSCs. In aging and diabetes, there were only mild changes with <15 dysregulated genes. The constitutive cell types that accomplish homeostatic renewal were conserved in DED but were associated with activation of cell states that comprise "adaptive regeneration." We provide global markers that distinguish cell types in homeostatic renewal vs. adaptive regeneration and markers that specifically define DED-elicited proliferating and differentiating cell types. We validate that expression of SPARC, a marker of adaptive regeneration, is also induced in corneal epithelial wound healing and accelerates wound closure in a corneal epithelial cell scratch assay. Finally, we propose a classification system for LSC markers based on their expression fidelity in homeostasis and disease. This transcriptional dissection uncovers the dramatically altered transcriptional landscape of the corneal epithelium in DED, providing a framework and atlas for future study of these ocular surface stem cells in health and disease.
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Síndromes do Olho Seco , Epitélio Corneano , Limbo da Córnea , Camundongos , Animais , Limbo da Córnea/fisiologia , Diferenciação Celular/fisiologia , Córnea , Cicatrização/genética , Síndromes do Olho Seco/genética , Síndromes do Olho Seco/metabolismo , Homeostase/genéticaRESUMO
RNA binding proteins are important regulators of T cell activation, proliferation, and cytokine production. The zinc finger protein 36 (ZFP36) family genes (Zfp36, Zfp36l1, and Zfp36l2) encode RNA binding proteins that promote the degradation of transcripts containing AU-rich elements. Numerous studies have demonstrated both individual and shared functions of the ZFP36 family in immune cells, but their collective function in T cells remains unclear. Here, we found a redundant and critical role for the ZFP36 proteins in regulating T cell quiescence. T cell-specific deletion of all three ZFP36 family members in mice resulted in early lethality, immune cell activation, and multiorgan pathology characterized by inflammation of the eyes, central nervous system, kidneys, and liver. Mice with T cell-specific deletion of any two Zfp36 genes were protected from this spontaneous syndrome. Triply deficient T cells overproduced proinflammatory cytokines, including IFN-γ, TNF, and GM-CSF, due to increased mRNA stability of these transcripts. Unexpectedly, T cell-specific deletion of both Zfp36l1 and Zfp36l2 rendered mice resistant to experimental autoimmune encephalomyelitits due to failed priming of antigen-specific CD4+ T cells. ZFP36L1 and ZFP36L2 double-deficient CD4+ T cells had poor proliferation during in vitro T helper cell polarization. Thus, the ZFP36 family redundantly regulates T cell quiescence at homeostasis, but ZFP36L1 and ZFP36L2 are specifically required for antigen-specific T cell clonal expansion.
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Fator Estimulador de Colônias de Granulócitos e Macrófagos , Linfócitos T , Tristetraprolina , Animais , Camundongos , Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Homeostase , Proteínas de Ligação a RNA/genética , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMO
Calcium regulates the response sensitivity, kinetics and adaptation in photoreceptors. In striped bass cones, this calcium feedback includes direct modulation of the transduction cyclic nucleotide-gated (CNG) channels by the calcium-binding protein CNG-modulin. However, the possible role of EML1, the mammalian homolog of CNG-modulin, in modulating phototransduction in mammalian photoreceptors has not been examined. Here, we used mice expressing mutant Eml1 to investigate its role in the development and function of mouse photoreceptors using immunostaining, in-vivo and ex-vivo retinal recordings, and single-cell suction recordings. We found that the mutation of Eml1 causes significant changes in the mouse retinal structure characterized by mislocalization of rods and cones in the inner retina. Consistent with the fraction of mislocalized photoreceptors, rod and cone-driven retina responses were reduced in the mutants. However, the Eml1 mutation had no effect on the dark-adapted responses of rods in the outer nuclear layer. Notably, we observed no changes in the cone sensitivity in the Eml1 mutant animals, either in darkness or during light adaptation, ruling out a role for EML1 in modulating cone CNG channels. Together, our results suggest that EML1 plays an important role in retina development but does not modulate phototransduction in mammalian rods and cones.
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Movimento Celular/genética , Sobrevivência Celular/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Animais , Cálcio/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Retina/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Visão Ocular/genéticaRESUMO
Macrophages undergo programmatic changes with age, leading to altered cytokine polarization and immune dysfunction, shifting these critical immune cells from protective sentinels to disease promoters. The molecular mechanisms underlying macrophage inflammaging are poorly understood. Using an unbiased RNA sequencing (RNA-seq) approach, we identified Mir146b as a microRNA whose expression progressively and unidirectionally declined with age in thioglycollate-elicited murine macrophages. Mir146b deficiency led to altered macrophage cytokine expression and reduced mitochondrial metabolic activity, two hallmarks of cellular aging. Single-cell RNA-seq identified patterns of altered inflammation and interferon gamma signaling in Mir146b-deficient macrophages. Identification of Mir146b as a potential regulator of macrophage aging provides novel insights into immune dysfunction associated with aging.
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Envelhecimento , Interferon gama/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos/fisiologia , MicroRNAs/metabolismo , Animais , Senescência Celular , Feminino , Expressão Gênica , Inflamação/metabolismo , Ativação de Macrófagos , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Mitocôndrias/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Tioglicolatos/farmacologiaRESUMO
PURPOSE: Growth Differentiation Factor 15 (GDF15) was previously identified as a molecular marker of retinal ganglion cell stress in rodent models of glaucoma and was elevated in the aqueous humor (AH) of patients with primary open-angle glaucoma as a possible risk factor for glaucoma progression. The purpose of this study was to determine whether changes in the AH GDF15 levels were associated with intraocular pressure (IOP) changes in eyes undergoing glaucoma surgery. METHODS: Here, we performed a prospective, longitudinal pilot study in nine patients to determine whether changes in AH GDF15 levels from surgery to post-surgery follow-up were associated with IOP fluctuation. An initial AH sample was taken from the peripheral corneal paracentesis during planned glaucoma surgery, and a second sample was taken during an outpatient follow-up visit, approximately six months later. RESULTS: There was a statistically significant correlation between GDF15 fold change and IOP standard deviation (r = 0.87, P = 0.003), IOP range (r = 0.87, P = 0.003), and maximum IOP (r = 0.86, P = 0.003). There was no correlation between the GDF15 fold change and baseline IOP (r = 0.50, P = 0.17), final IOP (r = 0.038, P = 0.92), or mean IOP (r = 0.40, P = 0.28). CONCLUSION: Our findings in this pilot study suggest that longitudinal changes in AH GDF15 may be associated with IOP fluctuation during the postoperative period. Further studies are necessary to corroborate these findings in a larger patient population and to explore the possibility that AH GDF15 may be used not only to improve treatment algorithms but also as a surrogate endpoint in clinical trials.
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Here, we report our studies of immune-mediated regulation of Zika virus (ZIKV), herpes simplex virus 1 (HSV-1), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the human cornea. We find that ZIKV can be transmitted via corneal transplantation in mice. However, in human corneal explants, we report that ZIKV does not replicate efficiently and that SARS-CoV-2 does not replicate at all. Additionally, we demonstrate that type III interferon (IFN-λ) and its receptor (IFNλR1) are expressed in the corneal epithelium. Treatment of human corneal explants with IFN-λ, and treatment of mice with IFN-λ eye drops, upregulates antiviral interferon-stimulated genes. In human corneal explants, blockade of IFNλR1 enhances replication of ZIKV and HSV-1 but not SARS-CoV-2. In addition to an antiviral role for IFNλR1 in the cornea, our results suggest that the human cornea does not support SARS-CoV-2 infection despite expression of ACE2, a SARS-CoV-2 receptor, in the human corneal epithelium.
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Betacoronavirus/fisiologia , Córnea/virologia , Infecções por Coronavirus/transmissão , Herpesvirus Humano 1/fisiologia , Interferons/imunologia , Pneumonia Viral/transmissão , Zika virus/fisiologia , Animais , Betacoronavirus/imunologia , COVID-19 , Córnea/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Herpes Simples/imunologia , Herpes Simples/transmissão , Herpes Simples/virologia , Humanos , Camundongos , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , SARS-CoV-2 , Replicação Viral/fisiologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia , Interferon lambdaRESUMO
Purpose: To determine whether increased growth differentiation factor 15 (GDF15) in aqueous humor (AH) is associated with worse visual field loss in patients with pseudoexfoliative glaucoma (PXG). Methods: We recruited 12 patients (6 males, 6 females) with primary open-angle glaucoma (POAG) or PXG who were scheduled to undergo glaucoma surgery. AH was obtained from the initial peripheral paracentesis for the planned glaucoma surgery, and GDF15 levels were quantified with enzyme-linked immunosorbent assay by an investigator masked to clinical information. Humphrey visual field testing was performed as a part of routine care; results were obtained by reviewing the medical record. Results: AH GDF15 was detectable in patients with POAG and PXG. Increased AH GDF15 was significantly associated with worse mean deviation in patients with POAG (r = -0.94; 95% confidence interval [CI], -0.99 to -0.33; P = 0.02) and PXG (r = -0.92; 95% CI, -0.99 to -0.41; P = 0.01). Conclusions: AH GDF15 is detectable in patients with PXG and POAG. Elevated AH GDF15 is strongly associated with worse mean deviation in both subgroups. These findings suggest that GDF15 may be a molecular marker of glaucoma severity that is generalizable to multiple types of glaucoma regardless of the underlying etiology. Translational Relevance: This study provides proof of concept that GDF15, a molecular marker of retinal ganglion stress that was initially identified in rodent models, may have clinical utility as a measure of glaucoma severity not only in POAG but also in PXG.
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Síndrome de Exfoliação , Glaucoma , Fator 15 de Diferenciação de Crescimento , Transtornos da Visão , Humor Aquoso , Síndrome de Exfoliação/diagnóstico , Feminino , Humanos , Masculino , Campos VisuaisRESUMO
Cell-autonomous endothelial cell (EC) fibroblast growth factor receptor (FGFR) signaling through FGFR1/2 is essential for injury-induced wound vascularization and pathologic neovascularization as in blinding eye diseases such as age-related macular degeneration. Which FGF ligand(s) is critical in regulating angiogenesis is unknown. Utilizing ex vivo models of choroidal endothelial sprouting and in vivo models of choroidal neovascularization (CNV), we demonstrate here that only FGF2 is the essential ligand. Though FGF-FGFR signaling can activate multiple intracellular signaling pathways, we show that FGF2 regulates pathogenic angiogenesis via STAT3 activation. The identification of FGF2 as a critical mediator in aberrant neovascularization provides a new opportunity for developing multi-target therapies in blinding eye diseases especially given the limitations of anti-VEGF monotherapy.
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Corioide/irrigação sanguínea , Neovascularização de Coroide/etiologia , Células Endoteliais/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator de Transcrição STAT3/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Western Blotting , Proliferação de Células , Células Cultivadas , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Injeções Intravítreas , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Advanced age-related macular degeneration (AMD), the leading cause of blindness among people over 50 years of age, is characterized by atrophic neurodegeneration or pathologic angiogenesis. Early AMD is characterized by extracellular cholesterol-rich deposits underneath the retinal pigment epithelium (RPE) called drusen or in the subretinal space called subretinal drusenoid deposits (SDD) that drive disease progression. However, mechanisms of drusen and SDD biogenesis remain poorly understood. Although human AMD is characterized by abnormalities in cholesterol homeostasis and shares phenotypic features with atherosclerosis, it is unclear whether systemic immunity or local tissue metabolism regulates this homeostasis. Here, we demonstrate that targeted deletion of macrophage cholesterol ABC transporters A1 (ABCA1) and -G1 (ABCG1) leads to age-associated extracellular cholesterol-rich deposits underneath the neurosensory retina similar to SDD seen in early human AMD. These mice also develop impaired dark adaptation, a cardinal feature of RPE cell dysfunction seen in human AMD patients even before central vision is affected. Subretinal deposits in these mice progressively worsen with age, with concomitant accumulation of cholesterol metabolites including several oxysterols and cholesterol esters causing lipotoxicity that manifests as photoreceptor dysfunction and neurodegeneration. These findings suggest that impaired macrophage cholesterol transport initiates several key elements of early human AMD, demonstrating the importance of systemic immunity and aging in promoting disease manifestation. Polymorphisms in genes involved with cholesterol transport and homeostasis are associated with a significantly higher risk of developing AMD, thus making these studies translationally relevant by identifying potential targets for therapy.
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Cegueira/induzido quimicamente , Cegueira/metabolismo , Colesterol/metabolismo , Degeneração Macular/induzido quimicamente , Degeneração Macular/metabolismo , Monócitos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Cegueira/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Ésteres do Colesterol/metabolismo , Progressão da Doença , Deleção de Genes , Humanos , Imunidade Inata , Degeneração Macular/imunologia , Degeneração Macular/patologia , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Oxisteróis/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Receptores Acoplados a Proteínas G/metabolismo , Retina/anormalidades , Retina/metabolismo , Retina/patologia , Epitélio Pigmentado da Retina/anormalidades , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologiaRESUMO
Photoreceptors have high intrinsic metabolic demand and are exquisitely sensitive to metabolic perturbation. In addition, they shed a large portion of their outer segment lipid membranes in a circadian manner, increasing the metabolic burden on the outer retina associated with the resynthesis of cell membranes and disposal of the cellular cargo. Here, we demonstrate that deletion of both ABCA1 and ABCG1 in rod photoreceptors leads to age-related accumulation of cholesterol metabolites in the outer retina, photoreceptor dysfunction, degeneration of rod outer segments, and ultimately blindness. A high-fat diet significantly accelerates rod neurodegeneration and vision loss, further highlighting the role of lipid homeostasis in regulating photoreceptor neurodegeneration and vision.
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Envelhecimento/metabolismo , Colesterol/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Transportador 1 de Cassete de Ligação de ATP/deficiência , Transportador 1 de Cassete de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/deficiência , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Envelhecimento/patologia , Envelhecimento/fisiologia , Animais , Deleção de Genes , Camundongos , Visão OcularRESUMO
Perturbations in WNT signaling are associated with congenital eye disorders, including familial exudative vitreoretinopathy and Norrie disease. More recently, activation of the WNT pathway has also been shown to be associated with age-related macular degeneration (AMD). In this study, we identified that in choroidal neovascular membranes from AMD patients, ß-catenin is activated specifically in the vascular endothelium, suggesting that WNT promotes pathologic angiogenesis by directly affecting vascular endothelial cells. WNT7B has been shown to be important during eye development for regression of the fetal hyaloid vasculature. However, it has not yet been established whether WNT7A and/or WNT7B are involved in neovascular AMD pathogenesis. Here, we show that WNT7A and WNT7B increase the proliferation of human dermal microvascular endothelial cells in a dose-dependent manner. Both WNT7A and WNT7B also stimulated vascular sprouting from mouse choroidal explants in vitro. To evaluate in vivo relevance, we generated mice systemically deficient in Wnt7a and/or Wnt7b. Genetic deletion of both Wnt7a and Wnt7b decreased the severity of laser injury-induced choroidal neovascularization (CNV), while individual deletion of either Wnt7a or Wnt7b did not have a significant effect on CNV, suggesting that WNT7A and WNT7B have redundant pro-angiogenic roles in vivo. Cumulatively, these findings identify specific WNT isoforms that may play a pathologic role in CNV as observed in patients with neovascular AMD. Although the source of increased WNT7A and/or WNT7B in CNV requires further investigation, WNT signaling may be a potential target for therapeutic intervention if these results are demonstrated to be relevant in human disease.
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Neovascularização de Coroide/metabolismo , Proteínas Wnt/fisiologia , Inibidores da Angiogênese/metabolismo , Animais , Proliferação de Células/fisiologia , Neovascularização de Coroide/patologia , Células Endoteliais/patologia , Humanos , Masculino , Camundongos , Transdução de Sinais/fisiologia , beta Catenina/metabolismoRESUMO
Macrophage aging is pathogenic in numerous diseases, including age-related macular degeneration (AMD), a leading cause of blindness in older adults. Although prior studies have explored the functional consequences of macrophage aging, less is known about its cellular basis or what defines the transition from physiologic aging to disease. Here, we show that despite their frequent self-renewal, macrophages from old mice exhibited numerous signs of aging, such as impaired oxidative respiration. Transcriptomic profiling of aged murine macrophages revealed dysregulation of diverse cellular pathways, especially in cholesterol homeostasis, that manifested in altered oxysterol signatures. Although the levels of numerous oxysterols in human peripheral blood mononuclear cells and plasma exhibited age-associated changes, plasma 24-hydroxycholesterol levels were specifically associated with AMD. These novel findings demonstrate that oxysterol levels can discriminate disease from physiologic aging. Furthermore, modulation of cholesterol homeostasis may be a novel strategy for treating age-associated diseases in which macrophage aging is pathogenic.
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Envelhecimento/sangue , Colesterol/sangue , Degeneração Macular/sangue , Oxisteróis/sangue , Envelhecimento/patologia , Animais , Modelos Animais de Doenças , Humanos , Leucócitos Mononucleares/patologia , Metabolismo dos Lipídeos/fisiologia , Macrófagos/metabolismo , Macrófagos/patologia , Degeneração Macular/patologia , Camundongos , Estresse OxidativoRESUMO
Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring 1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite 2 . Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines 2 , including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase2,3, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-IκBζ regulatory axis could be an important new strategy for the treatment of IL-17-IκBζ-mediated autoimmune diseases.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Proteínas I-kappa B/metabolismo , Succinatos/metabolismo , Animais , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-6/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , Psoríase/tratamento farmacológico , Psoríase/patologia , Estresse Fisiológico/efeitos dos fármacos , Succinatos/administração & dosagem , Succinatos/química , Succinatos/farmacologia , Succinatos/uso terapêutico , Receptores Toll-Like/imunologiaRESUMO
Glaucoma is the second leading cause of blindness worldwide. Physicians often use surrogate endpoints to monitor the progression of glaucomatous neurodegeneration. These approaches are limited in their ability to quantify disease severity and progression due to inherent subjectivity, unreliability, and limitations of normative databases. Therefore, there is a critical need to identify specific molecular markers that predict or measure glaucomatous neurodegeneration. Here, we demonstrate that growth differentiation factor 15 (GDF15) is associated with retinal ganglion cell death. Gdf15 expression in the retina is specifically increased after acute injury to retinal ganglion cell axons and in a murine chronic glaucoma model. We also demonstrate that the ganglion cell layer may be one of the sources of secreted GDF15 and that GDF15 diffuses to and can be detected in aqueous humor (AH). In validating these findings in human patients with glaucoma, we find not only that GDF15 is increased in AH of patients with primary open angle glaucoma (POAG), but also that elevated GDF15 levels are significantly associated with worse functional outcomes in glaucoma patients, as measured by visual field testing. Thus, GDF15 maybe a reliable metric of glaucomatous neurodegeneration, although further prospective validation studies will be necessary to determine if GDF15 can be used in clinical practice.