RESUMO
Significant association signals from genome-wide association studies (GWAS) point to genomic regions of interest. However, for most loci the causative genetic variant remains undefined. Determining expression quantitative trait loci (eQTL) in a disease relevant tissue is an excellent approach to zoom in on disease- or trait-associated association signals and hitherto on relevant disease mechanisms. To this end, we explored regulation of gene expression in healthy retina (n = 311) and generated the largest cis-eQTL data set available to date. Genotype- and RNA-Seq data underwent rigorous quality control protocols before FastQTL was applied to assess the influence of genetic markers on local (cis) gene expression. Our analysis identified 403,151 significant eQTL variants (eVariants) that regulate 3,007 genes (eGenes) (Q-Value < 0.05). A conditional analysis revealed 744 independent secondary eQTL signals for 598 of the 3,007 eGenes. Interestingly, 99,165 (24.71%) of all unique eVariants regulate the expression of more than one eGene. Filtering the dataset for eVariants regulating three or more eGenes revealed 96 potential regulatory clusters. Of these, 31 harbour 130 genes which are partially regulated by the same genetic signal. To correlate eQTL and association signals, GWAS data from twelve complex eye diseases or traits were included and resulted in identification of 80 eGenes with potential association. Remarkably, expression of 10 genes is regulated by eVariants associated with multiple eye diseases or traits. In conclusion, we generated a unique catalogue of gene expression regulation in healthy retinal tissue and applied this resource to identify potentially pleiotropic effects in highly prevalent human eye diseases. Our study provides an excellent basis to further explore mechanisms of various retinal disease etiologies.
Assuntos
Retina/metabolismo , Retina/fisiologia , Doenças Retinianas/genética , Autopsia , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Genótipo , Voluntários Saudáveis , Humanos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genéticaRESUMO
BACKGROUND: Dysfunctional humoral and cellular innate immunity are key components in the development and progression of age-related macular degeneration (AMD). Specifically, chronically activated microglia and their disturbed regulatory system contribute to retinal degeneration. Galectin-3, a ß-galactose binding protein, is a potent driver of macrophage and microglia activation and has been implicated in neuroinflammation, including neurodegenerative diseases of the brain. Here, we hypothesized that genetic deficiency of galectin-3 or its modulation via TD139 dampens mononuclear phagocyte reactivity and delays retinal degeneration. METHODS: Galectin-3 expression in AMD patients was analyzed by immunohistochemical stainings. Galectin-3 knockout and BALB/cJ mice were exposed to white bright light with an intensity of 15,000 lux for 1 h and Cx3cr1GFP/+ mice to focal blue light of 50,000 lux for 10 min. BALB/cJ and Cx3cr1GFP/+ mice received intraperitoneal injections of 15 mg/kg TD139 or vehicle for five consecutive days, starting one day prior to light exposure. The effects of galectin-3 deficiency or inhibition on microglia were analyzed by immunohistochemical stainings and in situ hybridization of retinal sections and flat mounts. Pro-inflammatory cytokine levels in the retina and retinal pigment epithelium (RPE) were quantified by qRT-PCR and transcriptomic changes were analyzed by RNA-sequencing. Retinal thickness and structure were evaluated by optical coherence tomography. RESULTS: We found that galectin-3 expression was strongly upregulated in reactive retinal mononuclear phagocytes of AMD patients and in the two related mouse models of light-induced retinal degeneration. The experimental in vivo data further showed that specific targeting of galectin-3 by genetic knockout or administration of the small-molecule inhibitor TD139 reduced microglia reactivity and delayed retinal damage in both light damage conditions. CONCLUSION: This study defines galectin-3 as a potent driver of retinal degeneration and highlights the protein as a drug target for ocular immunomodulatory therapies.
Assuntos
Galectina 3 , Degeneração Macular , Microglia , Animais , Citocinas/metabolismo , Galectina 3/antagonistas & inibidores , Galectina 3/genética , Galectina 3/metabolismo , Humanos , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/prevenção & controle , Camundongos , Microglia/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , RNA/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/prevenção & controle , Tiogalactosídeos/farmacologia , Triazóis/farmacologiaRESUMO
Retinal degeneration is a leading cause of visual impairment and blindness worldwide. Microglia reactivity is a hallmark of neurodegenerative diseases and a driving force for retinal cell death and disease progression. Thus, immunomodulation emerges as a potential therapeutic option. AhR deficiency is known to trigger inflammation and previous studies revealed important roles for AhR ligands in neuroprotection without focusing on microglia. Here, we investigate the anti-inflammatory and antioxidant effects of the synthetic aryl hydrocarbon receptor (AhR) ligand 2, 2'-aminophenyl indole (2AI) on microglia reactivity. We showed that 2AI potently reduced pro-inflammatory gene expression and induced antioxidant genes in activated human and murine microglia cells, in LPS-stimulated retinal explants as well as in stressed human ARPE-19 cells. 2AI also diminished LPS-induced nitric oxide (NO) release, their neurotoxic activity on photoreceptor cells, phagocytosis, and migration in murine BV-2 cells as important functional microglia parameters. siRNA-mediated knockdown of AhR partially prevented the previously observed gene regulatory effects in BV-2 cells. Our results show for the first time, that the synthetic AhR agonist 2AI regulates microglia homeostasis, highlighting AhR as a potential drug target for immunomodulatory and antioxidant therapies.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Indóis/química , Inflamação/metabolismo , Microglia/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Animais , Antioxidantes/metabolismo , Linhagem Celular , Movimento Celular , Inativação Gênica , Homeostase , Humanos , Ligantes , Lipopolissacarídeos/química , Camundongos , Óxido Nítrico/metabolismo , Nitritos , Fagocitose , Faloidina/química , RNA Interferente Pequeno/metabolismo , CicatrizaçãoRESUMO
Age-related macular degeneration (AMD) is a leading cause of visual impairment of the elderly population. Since AMD is a multifactorial age-related disease with various genetic risk factors, the understanding of its complex pathophysiology is still limited. However, animal experiments, genome-wide association data and the molecular profiling of AMD patient samples have highlighted a key role of systemic and local immune processes that contribute to this chronic eye disease. In this overview article, we concentrate on the role of lymphocytes and mononuclear phagocytes and their interplay in triggering a persistent immune response in the AMD retina. We preferentially review findings from human immune cell analyses and complement these with related findings in experimental models. We conclude that both immune cell types as their signaling network may be a rich source to identify novel molecular targets for immunomodulation in AMD.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Degeneração Macular/imunologia , Fagócitos/imunologia , Células Th1/imunologia , Células Th17/imunologia , Idoso de 80 Anos ou mais , Proteínas do Sistema Complemento/imunologia , Humanos , Imunomodulação/fisiologia , Degeneração Macular/patologia , Retina/imunologia , Retina/patologia , Transtornos da Visão/imunologiaRESUMO
Müller cells, the major retinal macroglia, are key to maintaining vascular integrity as well as retinal fluid and ion homeostasis. Although platelet derived growth factor (PDGF) receptor expression in Müller glia has been reported earlier, their actual role for Müller cell function and intimate interaction with cells of the retinal neurovascular unit remains unclear. To close this gap of knowledge, Müller cell-specific PDGF receptor alpha (PDGFRα) knockout (KO) mice were generated, characterized, and subjected to a model of choroidal neovascularization (CNV). PDGFRα-deficient Müller cells could not counterbalance hypoosmotic stress as efficiently as their wildtype counterparts. In wildtypes, the PDGFRα ligand PDGF-BB prevented Müller cell swelling induced by the administration of barium ions. This effect could be blocked by the PDGFR family inhibitor AC710. PDGF-BB could not restore the capability of an efficient volume regulation in PDGFRα KO Müller cells. Additionally, PDGFRα KO mice displayed reduced rod and cone-driven light responses. Altogether, these findings suggest that Müller glial PDGFRα is central for retinal functions under physiological conditions. In contrast, Müller cell-specific PDGFRα KO resulted in less vascular leakage and smaller lesion areas in the CNV model. Of note, the effect size was comparable to pharmacological blockade of PDGF signaling alone or in combination with anti-vascular endothelial growth factor (VEGF) therapy-a treatment regimen currently being tested in clinical trials. These data imply that targeting PDGF to treat retinal neovascular diseases may have short-term beneficial effects, but may elicit unwarranted side effects given the putative negative effects on Müller cell homeostatic functions potentially interfering with a long-term positive outcome.
Assuntos
Células Ependimogliais/metabolismo , Homeostase , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Imunofluorescência , Expressão Gênica , Técnicas de Inativação de Genes , Gliose/etiologia , Gliose/metabolismo , Gliose/patologia , Camundongos , Camundongos Knockout , Neuroglia/metabolismo , Especificidade de Órgãos/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Retina/metabolismo , Retina/patologiaRESUMO
Degeneration of the retinal pigment epithelium (RPE) is a hallmark of atrophic age-related macular degeneration (AMD). Microglia mediated inflammatory responses and oxidative stress are critical pathophysiological processes in the onset and progression of RPE degeneration. Given the central role of the RPE, strategies to protect these cells from damage caused by oxidative stress and inflammation present a promising therapeutic approach to mitigate AMD. Ligands for the translocator protein (18 kDa) (TSPO) have been shown to confer protection against retinal inflammatory responses and neurodegeneration by acting primarily through retinal glia. However, despite RPE cells demonstrating strong TSPO expression, it remains unclear whether TSPO ligands could also inhibit inflammatory responses of RPE cells. Here, we investigated the influence of three different TSPO ligands XBD173, PK11195 and Ro5-4864 on inflammatory responses in human ARPE-19 cells triggered by supernatants from reactive human microglial cells and the lysosomal destabilizer, LLOMe. Our findings revealed that TSPO ligands significantly inhibited proinflammatory gene expression, inflammasome-mediated caspase-1 activation, lipid accumulation and intracellular ROS levels in stressed ARPE-19 cells. Notably, TSPO ligands induced activation of Nrf2 pathway and its downstream regulated genes in ARPE-19 cells, with Hmox-1 being the most strongly upregulated gene. Collectively, our study indicates that TSPO ligands can enhance the Nrf2 antioxidant pathway in RPE cells and protect them from cellular damage resulting from inflammation and oxidative stress.
Assuntos
Inflamação/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Receptores de GABA/metabolismo , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais , Actinas/metabolismo , Autofagia , Caspase 1/metabolismo , Linhagem Celular , Citoesqueleto/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Mediadores da Inflamação/metabolismo , Ligantes , Metabolismo dos Lipídeos , Lipídeos/química , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Estresse FisiológicoRESUMO
BACKGROUND: Retinal degenerative diseases significantly contribute to visual impairment and blindness. Microglia reactivity is a hallmark of neurodegenerative diseases including retinal cell death and immunomodulation emerges as a therapeutic option. Indole-3-carbinol (I3C) is a natural ligand of aryl hydrocarbon receptor (AhR), with potent immunomodulatory properties. Here, we hypothesized that I3C may inhibit microglia reactivity and exert neuroprotective effects in the light-damaged murine retina mimicking important immunological aspects of retinal degeneration. METHODS: BV-2 microglia were treated in vitro with I3C followed by lipopolysaccharide (LPS) stimulation to analyze pro-inflammatory and anti-oxidant responses by quantitative real-time PCR (qRT-PCR) and Western blots. Nitric oxide (NO) secretion, caspase 3/7 levels, phagocytosis rates, migration, and morphology were analyzed in control and AhR knockdown cells. I3C or vehicle was systemically applied to light-treated BALB/cJ mice as an experimental model of retinal degeneration. Pro-inflammatory and anti-oxidant responses in the retina were examined by qRT-PCR, ELISA, and Western blots. Immunohistochemical staining of retinal flat mounts and cryosections were performed. The retinal thickness and structure were evaluated by in vivo imaging using spectral domain-optical coherence tomography (SD-OCT). RESULTS: The in vitro data showed that I3C potently diminished LPS-induced pro-inflammatory gene expression of I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 and induced anti-oxidants gene levels of NQO1, HMOX1, and CAT1 in BV-2 cells. I3C also reduced LPS-induced NO secretion, phagocytosis, and migration as important functional microglia parameters. siRNA-mediated knockdown of AhR partially prevented the previously observed gene regulatory events. The in vivo experiments revealed that I3C treatment diminished light-damage induced I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 transcripts and also reduced CCL2, I-NOS, IL-1ß, p-NFkBp65 protein levels in mice. Moreover, I3C increased anti-oxidant NQO1 and HMOX1 protein levels in light-exposed retinas. Finally, I3C therapy prevented the accumulation of amoeboid microglia in the subretinal space and protected from retinal degeneration. CONCLUSIONS: The AhR ligand I3C potently counter-acts microgliosis and light-induced retinal damage, highlighting a potential treatment concept for retinal degeneration.
Assuntos
Indóis/farmacologia , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Retina/efeitos dos fármacos , Degeneração Retiniana/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Feminino , Homeostase/efeitos dos fármacos , Indóis/uso terapêutico , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/patologia , Lipopolissacarídeos , Masculino , Camundongos , Microglia/patologia , Fármacos Neuroprotetores/uso terapêutico , Fagocitose/efeitos dos fármacos , Retina/patologiaRESUMO
INTRODUCTION: We present a prediction model for progression from early/intermediate to advanced age-related macular degeneration (AMD) within 5.9 years. OBJECTIVES: To evaluate the combined role of genetic, nongenetic, and phenotypic risk factors for conversion from early to late AMD over ≥5 years. METHODS: Baseline phenotypic characteristics were evaluated based on color fundus photography, spectral-domain optical coherence tomography, and infrared images. Genotyping for 36 single-nucleotide polymorphisms as well as systemic lipid and complement measurements were performed. Multivariable backward logistic regression resulted in a final prediction model. RESULTS AND CONCLUSIONS: During a mean of 5.9 years of follow-up, 22.4% (n = 52) of the patients (n = 232) showed progression to late AMD. The multivariable prediction model included age, CFH variant rs1061170, pigment abnormalities, drusenoid pigment epithelial detachment (DPED), and hyperreflective foci (HRF). The model showed an area under the curve of 0.969 (95% confidence interval 0.948-0.990) and adequate calibration (Hosmer-Lemeshow test, p = 0.797). In addition to advanced age and carrying a CFH variant, pigment abnormalities, DPED, and HRF are relevant imaging biomarkers for conversion to late AMD. In clinical routine, an intensified monitoring of patients with a high-risk phenotypic profile may be suitable for the early detection of conversion to late AMD.
Assuntos
Degeneração Macular , Descolamento Retiniano , Drusas Retinianas , Angiofluoresceinografia , Humanos , Degeneração Macular/diagnóstico por imagem , Tomografia de Coerência ÓpticaRESUMO
Choroidal neovascularization (CNV) is a pathological process in which aberrant blood vessels invade the subretinal space of the mammalian eye. It is a characteristic feature of the prevalent neovascular age-related macular degeneration (nAMD). Circulating microRNAs (cmiRNAs) are regarded as potentially valuable biomarkers for various age-related diseases, including nAMD. Here, we investigated cmiRNA expression in an established laser-induced CNV mouse model. Upon CNV induction in C57Bl/6 mice, blood-derived cmiRNAs were initially determined globally by RNA next generation sequencing, and the most strongly dysregulated cmiRNAs were independently replicated by quantitative reverse transcription PCR (RT-qPCR) in blood, retinal, and retinal pigment epithelium (RPE)/choroidal tissue. Our findings suggest that two miRNAs, mmu-mir-486a-5p and mmur-mir-92a-3p, are consistently dysregulated during CNV formation. Furthermore, in functional in vitro assays, a significant impact of mmu-mir-486a-5p and mmu-mir-92a-3p on murine microglial cell viability was observed, while mmu-mir-92a-3p also showed an impact on microglial mobility. Taken together, we report a robust dysregulation of two miRNAs in blood and RPE/choroid after laser-induced initiation of CNV lesions in mice, highlighting their potential role in pathology and eventual therapy of CNV-associated complications.
Assuntos
Neovascularização de Coroide/sangue , Neovascularização de Coroide/etiologia , MicroRNA Circulante/genética , Lasers/efeitos adversos , Animais , Células Cultivadas , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Células Endoteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , MicroRNAs/genética , Microglia/metabolismo , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , TranscriptomaRESUMO
Inherited retinal dystrophies (iRDs) are a group of genetically and clinically heterogeneous conditions resulting from mutations in over 250 genes. Here, homozygosity mapping and whole-exome sequencing (WES) in a consanguineous family revealed a homozygous missense mutation, c.973C>T (p.His325Tyr), in RCBTB1. In affected individuals, it was found to segregate with retinitis pigmentosa (RP), goiter, primary ovarian insufficiency, and mild intellectual disability. Subsequent analysis of WES data in different cohorts uncovered four additional homozygous missense mutations in five unrelated families in whom iRD segregates with or without syndromic features. Ocular phenotypes ranged from typical RP starting in the second decade to chorioretinal dystrophy with a later age of onset. The five missense mutations affect highly conserved residues either in the sixth repeat of the RCC1 domain or in the BTB1 domain. A founder haplotype was identified for mutation c.919G>A (p.Val307Met), occurring in two families of Mediterranean origin. We showed ubiquitous mRNA expression of RCBTB1 and demonstrated predominant RCBTB1 localization in human inner retina. RCBTB1 was very recently shown to be involved in ubiquitination, more specifically as a CUL3 substrate adaptor. Therefore, the effect on different components of the CUL3 and NFE2L2 (NRF2) pathway was assessed in affected individuals' lymphocytes, revealing decreased mRNA expression of NFE2L2 and several NFE2L2 target genes. In conclusion, our study puts forward mutations in RCBTB1 as a cause of autosomal-recessive non-syndromic and syndromic iRD. Finally, our data support a role for impaired ubiquitination in the pathogenetic mechanism of RCBTB1 mutations.
Assuntos
Alelos , Fatores de Troca do Nucleotídeo Guanina/genética , Mutação de Sentido Incorreto/genética , Distrofias Retinianas/genética , Ubiquitinação/genética , Adolescente , Adulto , Idade de Início , Criança , Consanguinidade , Proteínas Culina/metabolismo , Exoma/genética , Feminino , Efeito Fundador , Genes Recessivos , Haplótipos/genética , Homozigoto , Humanos , Linfócitos/metabolismo , Masculino , Fator 2 Relacionado a NF-E2/metabolismo , Linhagem , Fenótipo , RNA Mensageiro/genética , Retina/metabolismo , Síndrome , TurquiaRESUMO
BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of visual impairment in the elderly. The neovascular (wet) form of AMD can be treated with intravitreal injections of different anti-vascular endothelial growth factor (VEGF) agents. Placental growth factor (PGF) is another member of the VEGF family of cytokines with pro-angiogenic and pro-inflammatory effects. Here, we aimed to compare single and combined inhibition of VEGF-A and PGF in the laser-induced mouse model of choroidal neovascularization (CNV) with a focus on the effects on retinal mononuclear phagocytes. METHODS: CNV was induced in C57BL/6J mice using a YAG-Laser. Immediately after laser damage antibodies against VEGF-A (aVEGF), anti-PGF (aPGF), aVEGF combined with aPGF, aflibercept, or IgG control were injected intravitreally in both eyes. Three and 7 days after laser damage, the vascular leakage was determined by fluorescence angiography. Lectin staining of retinal and RPE/choroidal flat mounts was used to monitor CNV. In situ mRNA co-expression of Iba1, VEGF and PGF were quantified using in situ hybridization. Retinal and RPE/choroidal protein levels of VEGF and PGF as well as the pro-inflammatory cytokines IL-6, IL1-beta, and TNF were determined by ELISA. RESULTS: Early (day 3) and intermediate (day 7) vascular leakage and CNV were significantly inhibited by PGF and VEGF-A co-inhibition, most effectively with the trap molecule aflibercept. While VEGF-A blockage alone had no effects, trapping PGF especially with aflibercept prevented the accumulation of reactive microglia and macrophages in laser lesions. The lesion-related mRNA expression and secretion of VEGF-A and PGF by mononuclear phagocytes were potently suppressed by PGF and partially by VEGF-A inhibition. Protein levels of IL-6 and IL1-beta were strongly reduced in all treatment groups. CONCLUSIONS: Retinal inhibition of PGF in combination with VEGF-A prevents vascular leakage and CNV possibly via modulating their own expression in mononuclear phagocytes. PGF-related, optimized strategies to target inflammation-mediated angiogenesis may help to increase efficacy and reduce non-responders in the treatment of wet AMD patients.
Assuntos
Monócitos/metabolismo , Neovascularização Patológica/prevenção & controle , Fator de Crescimento Placentário/antagonistas & inibidores , Doenças Retinianas/prevenção & controle , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Plexo Corióideo/patologia , Citocinas/metabolismo , Feminino , Interleucina-1/antagonistas & inibidores , Interleucina-1/biossíntese , Interleucina-6/antagonistas & inibidores , Interleucina-6/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Neovascularização Patológica/patologia , Fator de Crescimento Placentário/biossíntese , RNA Mensageiro/biossíntese , Retina/patologia , Doenças Retinianas/patologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
The original version of this Article contained an error in the spelling of the author Anja K. Mayer, which was incorrectly given as Anja Kathrin Mayer. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMO
Translocator protein (18â¯kDa) (TSPO) is a mitochondrial protein expressed by reactive microglia and astrocytes at the site of neuronal injury. Although TSPO function has not been fully determined, synthetic TSPO ligands have beneficial effects on different pathologies of the central nervous system, including the retina. Here, we studied the pattern of Tspo expression in the aging human retina and in two mouse models of retinal degeneration. Using a newly generated Tspo-KO mouse, we investigated the impact of the lack of TSPO on retinal morphology, function and susceptibility to degeneration. We show that TSPO was expressed in both human and mouse retina and retinal pigment epithelium (RPE). Tspo was induced in the mouse retina upon degeneration, but constitutively expressed in the RPE. Similarly, TSPO expression levels in healthy human retina and RPE were not differentially regulated during aging. Tspo-KO mice had normal retinal morphology and function up to 48 weeks of age. Photoreceptor loss caused either by exposure to excessive light levels or by a mutation in the phosphodiesterase 6b gene was not affected by the absence of Tspo. The reactivity states of retinal mononuclear phagocytes following light-damage were comparable in Tspo-KO and control mice. Our data suggest that lack of endogenous TSPO does not directly influence the magnitude of photoreceptor degeneration or microglia activation in these two models of retinal degeneration. We therefore hypothesize that the interaction of TSPO with its ligands may be required to modulate disease progression.
Assuntos
Regulação da Expressão Gênica/fisiologia , Receptores de GABA/genética , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Eletrorretinografia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Pessoa de Meia-Idade , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/patologia , Tomografia de Coerência ÓpticaRESUMO
Genetic variants of high-temperature requirement A serine peptidase 1 (HTRA1) and age-related maculopathy susceptibility 2 (ARMS2) are associated with age-related macular degeneration (AMD). One HTRA1 single nucleotide polymorphism (SNP) is situated in the promotor region (rs11200638) resulting in increased expression, while two synonymous SNPs are located in exon 1 (rs1049331:C > T, rs2293870:G > T). HtrA1 is known to inhibit transforming growth factor-ß (TGF-ß) signaling, a pathway regulating quiescence of microglia, the resident immune cells of the brain and retina. Microglia-mediated immune responses contribute to AMD pathogenesis. It is currently unclear whether AMD-associated HTRA1 variants influence TGF-ß signaling and microglia phenotypes. Here, we show that an HtrA1 isoform carrying AMD-associated SNPs in exon 1 exhibits increased proteolytic activity. However, when incubating TGF-ß-treated reactive microglia with HtrA1 protein variants, neither the wildtype nor the SNP-associated isoforms changed microglia activation in vitro.
Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Degeneração Macular/genética , Microglia/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Predisposição Genética para Doença , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único , Serina EndopeptidasesRESUMO
Mattapallil et al. described that vendor lines for C57BL/6 N mice may carry the rd8 mutation that leads to an ocular phenotype, which could be mistaken for an induced retinal degeneration. This mouse strain is widely used in ophthalmic research as a background for modeling retinal degeneration. In the process of studying Cln3Δex7/8 knock-in mice on a C57BL/6 N background, we became aware of this issue. The aim of this study thus was to use electroretinography to investigate the age-dependent functional loss in Cln3+/+ rd8-/rd8- mice and compare it to C57BL/6 J mice.The scotopic and photopic amplitudes of the a-wave and b-wave decrease significantly in mutant mice with increasing age, and the implicit time is prolonged. Especially the oscillatory potentials arising from inner retinal interaction seem to be notably affected by the rd8 mutation. Surprisingly, the amplitudes in young C57BL/6 J mice were lower than those measured in C57BL/6 N at any time point.Our results indicate that the rd8 mutation present in C57BL/6 N mice affects the function of the inner and outer retina. This is surprising given that the major retinal morphological alterations due to the rd8 mutation are found in the outer retina.We conclude that the rd8 mutation does affect the retinal function in Cln3+/+ rd8-/rd8- mice in a variable manner. Epigenetic factors and modifying genes lead to a phenotype shift in these mice. Interpreting the results of previous studies in mutant mice on C57BL/6 N background is challenging as comparing results obtained in independent studies or on other mouse backgrounds may be misleading. Using littermates as controls remains the only valid option.
Assuntos
Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Degeneração Retiniana/genética , Envelhecimento , Animais , Modelos Animais de Doenças , Eletrorretinografia , Técnicas de Introdução de Genes , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Retina/fisiopatologiaRESUMO
Microglia activation is central to the pathophysiology of retinal degenerative disorders. Resveratrol, a naturally occurring non-flavonoid phenolic compound present in red wine has potent anti-inflammatory and immunomodulatory properties. However, molecular mechanisms by which resveratrol influences microglial inflammatory pathways and housekeeping functions remain unclear. Here, we first studied the immuno-modulatory effects of resveratrol on BV-2 microglial cells at the transcriptome level using DNA-microarrays and selected qRT-PCR analyses. We then analyzed resveratrol effects on microglia morphology, phagocytosis and migration and estimated their neurotoxicity on 661â¯W photoreceptors by quantification of caspase 3/7 levels. We found that resveratrol effectively blocked gene expression of a broad spectrum of lipopolysaccharide (LPS)-induced pro-inflammatory molecules, including cytokines and complement proteins. These transcriptomic changes were accompanied by potent inhibition of LPS-induced nitric oxide secretion and reduced microglia-mediated apoptosis of 661â¯W photoreceptor cultures. Our findings highlight novel targets involved in the anti-inflammatory and neuroprotective action of resveratrol against neuroinflammatory responses.
Assuntos
Microglia/efeitos dos fármacos , Microglia/metabolismo , Estilbenos/farmacologia , Transcriptoma/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Camundongos , Microglia/citologia , Modelos Neurológicos , Degeneração Neural/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fagocitose/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real , ResveratrolRESUMO
PurposePart of the hidden genetic variation in heterogeneous genetic conditions such as inherited retinal diseases (IRDs) can be explained by copy-number variations (CNVs). Here, we explored the genomic landscape of IRD genes listed in RetNet to identify and prioritize those genes susceptible to CNV formation.MethodsRetNet genes underwent an assessment of genomic features and of CNV occurrence in the Database of Genomic Variants and literature. CNVs identified in an IRD cohort were characterized using targeted locus amplification (TLA) on extracted genomic DNA.ResultsExhaustive literature mining revealed 1,345 reported CNVs in 81 different IRD genes. Correlation analysis between rankings of genomic features and CNV occurrence demonstrated the strongest correlation between gene size and CNV occurrence of IRD genes. Moreover, we identified and delineated 30 new CNVs in IRD cases, 13 of which are novel and three of which affect noncoding, putative cis-regulatory regions. Finally, the breakpoints of six complex CNVs were determined using TLA in a hypothesis-neutral manner.ConclusionWe propose a ranking of CNV-prone IRD genes and demonstrate the efficacy of TLA for the characterization of CNVs on extracted DNA. Finally, this IRD-oriented CNV study can serve as a paradigm for other genetically heterogeneous Mendelian diseases with hidden genetic variation.
Assuntos
Mapeamento Cromossômico , Variações do Número de Cópias de DNA , Genoma Humano , Genômica , Fases de Leitura Aberta , RNA não Traduzido , Doenças Retinianas/genética , Alelos , Proteínas Relacionadas a Caderinas , Caderinas/genética , Bases de Dados Genéticas , Proteínas do Olho/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genômica/métodos , Humanos , Sequências Reguladoras de Ácido Nucleico , Doenças Retinianas/diagnóstico , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
Neuronal ceroid lipofuscinosis (NCL) is the most common group of neurogenetic storage diseases typically beginning in childhood. The juvenile form (JNCL), also known as Batten disease, is the most common form. Vision-related problems are often an early sign, appearing prior to motor and mental deficits. We have previously investigated disease progression with age in the Cln3 Δex7/8 KI mouse model for JNCL and showed a decline of visual acuity and a predominant decline of the inner retinal function in mice, similar to human disease. The aim of this study was to further characterize this degeneration by means of flicker ERGs. For the scotopic flicker ERG, we found a significantly lower magnitude for Cln3 Δex7/8 KI mice already at 6 months of age for low stimulus frequencies, while the difference declines with increasing frequency. Under photopic conditions there was no magnitude difference at 6 months, but a cumulative magnitude reduction with further aging. For both conditions the phases were similar for both groups. There was a similar magnitude reduction for the responses of both the slow and fast rod pathway in the 15 Hz experiments, and there were no differences in response phase. Low-frequency flicker responses seem to be sensitive to very early disease manifestations, and while the degeneration is associated with a reduction of predominating inner retinal responses in the scotopic flash ERG, this predominance seems not to be related to a selective involvement of the slow and fast rod pathways.
Assuntos
Proteínas do Olho/genética , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Lipofuscinoses Ceroides Neuronais/fisiopatologia , Degeneração Retiniana/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Envelhecimento , Animais , Modelos Animais de Doenças , Eletrorretinografia , Proteínas do Olho/fisiologia , Junções Comunicantes/fisiologia , Técnicas de Introdução de Genes , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/fisiologia , Lipofuscinoses Ceroides Neuronais/genética , Visão Noturna , Degeneração Retiniana/genética , Vias Visuais/fisiologiaRESUMO
Photoreceptor-specific gene expression is controlled by a hierarchical network of transcription factors, including the master regulators cone-rod homeobox (Crx) and neural retina leucine zipper (Nrl). Myocyte-enhancer factor 2c (Mef2c) is an ubiquitously expressed transcription factor with important functions in the cardiovascular system. Here, we performed a detailed analysis of Mef2c expression, localization and function in the retina to further elucidate its potential role for photoreceptor gene regulation. We showed that murine retinal Mef2c mRNA expression was high at birth and peaked at late postnatal developmental stages. Using immunohistochemistry and Western blot, Mef2c protein was detected in the outer nuclear layer of adult mouse and human retinas and localized to the nucleus of 661W photoreceptor-like cells. Mef2c knock-down in 661W cells reduced the expression of arrestin 3 (Arr3) and medium-wave-sensitive cone opsin (Opn1mw) but increased transcript levels of mitogen-activated protein kinase 15 (Mapk15) and phosphodiesterase 6h (Pde6h). In conclusion, Mef2c is highly expressed in the retina where it modulates photoreceptor-specific gene expression.
Assuntos
Regulação da Expressão Gênica , Retina/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Animais , Arrestinas/metabolismo , Núcleo Celular/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/metabolismo , Distribuição TecidualRESUMO
Activation of the innate immune system plays a major role in retinal degenerative diseases including age-related macular degeneration (AMD). In this study, we investigated whether reactive microglia trigger and sustain NLRP3 inflammasome activation in human retinal pigment epithelium (ARPE-19) cells. Specifically, we analyzed the potential of cell culture supernatants from lipopolysaccharide (LPS)-stimulated human microglia in combination with the lysosomal destabilization agent Leu-Leu-O-Me (LLOMe) to activate the inflammasome in ARPE-19 cells. We found disorganization of ARPE-19 cytoskeletal structure after incubation with conditioned media of LPS-stimulated microglia and LLOMe and accumulation of lipid deposits in these cells using Nile Red staining. LC3-II, the active form of the autophagy marker microtubule-associated protein 1 light chain 3 beta (LC3B), was also elevated in ARPE-19 cells after inducing inflammasome activation. Finally, a significant increase of transcripts for IL-6, IL-8, IL-1ß, GM-CSF and CCL-2 was detected in ARPE-19 stimulated with both microglia-conditioned medium and LLOMe. Our findings highlight a potential role of microglia in RPE inflammasome activation.