RESUMEN
Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from Ciona intestinalis (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction.
Asunto(s)
Mitocondrias , Oxidorreductasas , Proteínas de Plantas , Epitelio Pigmentado de la Retina , Animales , Mitocondrias/metabolismo , Ratones , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Ciona intestinalis/metabolismo , Ubiquinona/metabolismo , Ubiquinona/análogos & derivados , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Degeneración Retiniana/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologíaRESUMEN
Greg Lemke's laboratory was one of the pioneers of research into the TAM family of receptor tyrosine kinases (RTKs). Not only was Tyro3 cloned in his laboratory, but his group also extensively studied mice knocked out for individual or various combinations of the TAM RTKs Tyro3, Axl, and Mertk. Here we primarily focus on one of the paralogs-MERTK. We provide a historical perspective on rodent models of loss of Mertk function and their association with retinal degeneration and blindness. We describe later studies employing mouse genetics and the generation of newer knockout models that point out incongruencies with the inference that loss of MERTK-dependent phagocytosis is sufficient for severe, early-onset photoreceptor degeneration in mice. This discussion is meant to raise awareness with regards to the limitations of the original Mertk knockout mouse model generated using 129 derived embryonic stem cells and carrying 129 derived alleles and the role of these alleles in modifying Mertk knockout phenotypes or even displaying Mertk-independent phenotypes. We also suggest molecular approaches that can further Greg Lemke's scintillating legacy of dissecting the molecular functions of MERTK-a protein that has been described to function in phagocytosis as well as in the negative regulation of inflammation.
Asunto(s)
Ratones Noqueados , Fagocitosis , Tirosina Quinasa c-Mer , Animales , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Ratones , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Modelos Animales de Enfermedad , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Humanos , Inflamación/genética , Inflamación/metabolismoRESUMEN
Cre-mediated modulation of gene function in the murine retinal pigment epithelium (RPE) has been widely used, but current postnatal RPE-selective Cre driver lines suffer from limited recombination efficiency and/or ectopic or mosaic expression. We sought to generate a transgenic mouse line with consistently efficient RPE-selective Cre activity that could be temporally regulated. We used ÏC31 integrase to insert a DNA construct encoding a human BEST1 promoter fragment driving a Cre recombinase estrogen receptor fusion (BEST1-CreERT2) at the Rosa26 locus of C57BL/6J mice. Rosa26BEST1-CreERT2 mice were bred with a tdTomato reporter line and to mice with a Cre-conditional allele of Tfam. 4-hydroxytamoxifen or vehicle was delivered by four consecutive daily intraperitoneal injections. TdTomato was robustly expressed in the RPE of mice of both sexes for inductions beginning at P14 (males 90.7 ± 4.5%, females 84.7 ± 3.2%) and at 7 weeks (males 84.3 ± 7.0%, females 82 ± 3.6%). <0.6% of Muller glia also expressed tdTomato, but no tdTomato fluorescence was observed in other ocular cells or in multiple non-ocular tissues, with the exception of sparse foci in the testis. No evidence of retinal toxicity was observed in mice homozygous for the transgene induced beginning at P14 and assessed at 7-10 months. RPE-selective ablation of Tfam beginning at P14 led to reduced retinal thickness at 8 months of age and diminished retinal electrical responses at 12 months, as expected. These findings demonstrate that we have generated a mouse line with consistently efficient, tamoxifen-mediated postnatal induction of Cre recombination in the RPE and a small fraction of Muller glia. This line should be useful for temporally regulated modulation of gene function in the murine RPE.
Asunto(s)
Regulación de la Expresión Génica , Integrasas/genética , Degeneración Macular/genética , Epitelio Pigmentado de la Retina/metabolismo , Animales , Modelos Animales de Enfermedad , Electrorretinografía , Integrasas/biosíntesis , Degeneración Macular/metabolismo , Degeneración Macular/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN/genética , ARN/metabolismo , Epitelio Pigmentado de la Retina/patología , Tomografía de Coherencia ÓpticaRESUMEN
Purpose: The Seahorse XFp platform is widely used for metabolic assessment of cultured cells. Current methods require replating of cells into specialized plates. This is problematic for certain cell types, such as primary human fetal RPE (hfRPE) cells, which must be cultured for months to become properly differentiated. Our goal was to overcome this limitation by devising a method for assaying intact cell monolayers with the Seahorse XFp, without the need for replating. Methods: Primary hfRPE cells were differentiated by prolonged culture on filter inserts. Triangular sections of filters with differentiated cells attached were excised, transferred to XFp cell culture miniplate wells, immobilized at the bottoms, and subjected to mitochondrial stress tests. Replated cells were measured for comparison. Differentiated hfRPE cells were challenged or not with bovine photoreceptor outer segments (POS), and mitochondrial stress tests were performed 3.5 h later, after filter excision and transfer to assay plates. Results: Differentiated hfRPE cells assayed following filter excision demonstrated increased maximal respiration, increased spare respiration capacity, and increased extracellular acidification rate (ECAR) relative to replated controls. hfRPE cells challenged with POS exhibited increased maximal respiration and spare capacity, with no apparent change in the ECAR, relative to untreated controls. Conclusions: We have developed a method to reproducibly assay intact, polarized monolayers of hfRPE cells with the Seahorse XFp platform and have shown that the method yields more robust metabolic measurements compared to standard methods and is suitable for assessing the consequences of prolonged perturbations of differentiated cells. We expect our approach to be useful for a variety of studies involving metabolic assessment of adherent cells cultured on filters.
Asunto(s)
Bioensayo , Respiración de la Célula/efectos de los fármacos , Células Inmovilizadas/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Pigmentos Retinianos/farmacología , Animales , Transporte Biológico , Bovinos , Diferenciación Celular , Respiración de la Célula/fisiología , Células Inmovilizadas/citología , Células Inmovilizadas/metabolismo , Cámaras de Difusión de Cultivos , Metabolismo Energético/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Feto , Humanos , Mitocondrias/metabolismo , Fagocitosis/fisiología , Cultivo Primario de Células , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Pigmentos Retinianos/aislamiento & purificaciónRESUMEN
Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of Mertk-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in Mertk-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. Mertk-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of Mertk. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in Mertk knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of Mertk knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates Mertk-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD.
Asunto(s)
Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Degeneración Retiniana/genética , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , Fagocitosis , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Retina/metabolismo , Retina/patología , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/patología , Tirosina Quinasa c-MerRESUMEN
Retinitis pigmentosa (RP), a disease characterized by progressive loss of photoreceptors, exhibits significant genetic heterogeneity. Several genes associated with U4/U6-U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex of the spliceosome have been implicated in autosomal dominant RP (adRP). HPrp4, encoded by PRPF4, regulates the stability of U4/U6 di-snRNP, which is essential for continuous splicing. Here, we identified two heterozygous variants in PRPF4, including c.-114_-97del in a simplex RP patient and c.C944T (p.Pro315Leu), which co-segregates with disease phenotype in a family with adRP. Both variants were absent in 400 unrelated controls. The c.-114_-97del, predicted to affect two transcription factor binding sites, was shown to down-regulate the promoter activity of PRPF4 by a luciferase assay, and was associated with a significant reduction of PRPF4 expression in the blood cells of the patient. In fibroblasts from an affected individual with the p.Pro315Leu variant, the expression levels of several tri-snRNP components, including PRPF4 itself, were up-regulated, with altered expression pattern of SC35, a spliceosome marker. The same alterations were also observed in cells over expressing hPrp4(Pro315Leu), suggesting that they arose as a compensatory response to a compromised splicing mechanism caused by hPrp4 dysfunction. Further, over expression of hPrp4(Pro315Leu), but not hPrp4(WT), triggered systemic deformities in wild-type zebrafish embryos with the retina primarily affected, and dramatically augmented death rates in morphant embryos, in which orthologous zebrafish prpf4 gene was silenced. We conclude that mutations of PRPF4 cause RP via haploinsufficiency and dominant-negative effects, and establish PRPF4 as a new U4/U6-U5 snRNP component associated with adRP.
Asunto(s)
Mutación Missense , Retinitis Pigmentosa/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Niño , Regulación hacia Abajo , Femenino , Genes Dominantes , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Regiones Promotoras Genéticas , Retinitis Pigmentosa/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/química , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Alineación de Secuencia , Adulto JovenRESUMEN
MERTK is an essential component of the signaling network that controls phagocytosis in retinal pigment epithelium (RPE), the loss of which results in photoreceptor degeneration. Previous proof-of-concept studies have demonstrated the efficacy of gene therapy using human MERTK (hMERTK) packaged into adeno-associated virus (AAV2) in treating RCS rats and mice with MERTK deficiency. The purpose of this study was to assess the safety of gene transfer via subretinal administration of rAAV2-VMD2-hMERTK in subjects with MERTK-associated retinitis pigmentosa (RP). After a preclinical phase confirming the safety of the study vector in monkeys, six patients (aged 14 to 54, mean 33.3 years) with MERTK-related RP and baseline visual acuity (VA) ranging from 20/50 to <20/6400 were entered in a phase I open-label, dose-escalation trial. One eye of each patient (the worse-seeing eye in five subjects) received a submacular injection of the viral vector, first at a dose of 150 µl (5.96 × 10(10)vg; 2 patients) and then 450 µl (17.88 × 10(10)vg; 4 patients). Patients were followed daily for 10 days at 30, 60, 90, 180, 270, 365, 540, and 730 days post-injection. Collected data included (1) full ophthalmologic examination including best-corrected VA, intraocular pressure, color fundus photographs, macular spectral domain optical coherence tomography and full-field stimulus threshold test (FST) in both the study and fellow eyes; (2) systemic safety data including CBC, liver and kidney function tests, coagulation profiles, urine analysis, AAV antibody titers, peripheral blood PCR and ASR measurement; and (3) listing of ophthalmological or systemic adverse effects. All patients completed the 2-year follow-up. Subretinal injection of rAAV2-VMD2-hMERTK was associated with acceptable ocular and systemic safety profiles based on 2-year follow-up. None of the patients developed complications that could be attributed to the gene vector with certainty. Postoperatively, one patient developed filamentary keratitis, and two patients developed progressive cataract. Of these two patients, one also developed transient subfoveal fluid after the injection as well as monocular oscillopsia. Two patients developed a rise in AAV antibodies, but neither patient was positive for rAAV vector genomes via PCR. Three patients also displayed measurable improved visual acuity in the treated eye following surgery, although the improvement was lost by 2 years in two of these patients. Gene therapy for MERTK-related RP using careful subretinal injection of rAAV2-VMD2-hMERTK is not associated with major side effects and may result in clinical improvement in a subset of patients.
Asunto(s)
Terapia Genética/métodos , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/terapia , Adolescente , Adulto , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Determinación de Punto Final , Femenino , Estudios de Seguimiento , Vectores Genéticos , Humanos , Macaca , Masculino , Persona de Mediana Edad , Mutación , Complicaciones Posoperatorias/terapia , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Líquido Subretiniano , Tomografía de Coherencia Óptica , Resultado del Tratamiento , Agudeza Visual , Adulto Joven , Tirosina Quinasa c-MerRESUMEN
Retinal pigment epithelium (RPE) cell migration in response to disease has been reported for age-related macular degeneration, proliferative vitreoretinopathy, and proliferative diabetic retinopathy. The complex molecular process of RPE cell migration is regulated in part by growth factors and cytokines, and activation of the PI3/AKT/mTOR signaling pathway. Rapamycin, an allosteric mTOR inhibitor, has been shown to block only one of the primary downstream mTOR effectors, p70 S6 kinase 1, in many cell types. INK128, a selective mTOR ATP binding site competitor, blocks both p70 S6 kinase 1 and a second primary downstream effector, 4E-BP1. We performed scratch assays using differentiated ARPE-19 and primary porcine RPE cells to assess the effect of mTOR inhibition on cell migration. We found that INK128-mediated blocking of both p70 S6 kinase 1 and 4E-BP1 was much more effective at preventing RPE cell migration than rapamycin-mediated inhibition of p70 S6 kinase 1 alone.
Asunto(s)
Benzoxazoles/farmacología , Movimiento Celular/efectos de los fármacos , Pirimidinas/farmacología , Epitelio Pigmentado de la Retina/citología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular , Línea Celular , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Immunoblotting , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Sirolimus/farmacología , Porcinos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
MERTK-associated retinal degenerations are thought to have defects in phagocytosis of shed outer segment membranes by the retinal pigment epithelium (RPE), as do the rodent models of these diseases. We have subretinally injected an RPE-specific AAV2 vector, AAV2-VMD2-hMERTK, to determine whether this would provide long-term photoreceptor rescue in the RCS rat, which it did for up to 6.5 months, the longest time point examined. Moreover, we found phagosomes in the RPE in the rescued regions of RCS retinas soon after the onset of light. The same vector also had a major protective effect in Mertk-null mice, with a concomitant increase in ERG response amplitudes in the vector-injected eyes. These findings suggest that planned clinical trials with this vector will have a favorable outcome.
Asunto(s)
Terapia Genética/métodos , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Degeneración Retiniana/genética , Degeneración Retiniana/terapia , Animales , Bestrofinas , Canales de Cloruro/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Electrorretinografía , Proteínas del Ojo/genética , Vectores Genéticos/genética , Humanos , Ratones Noqueados , Fagocitosis/genética , Fagocitosis/fisiología , Fagosomas/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/metabolismo , Ratas Mutantes , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/deficiencia , Proteínas Tirosina Quinasas Receptoras/metabolismo , Degeneración Retiniana/fisiopatología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/fisiopatología , Resultado del Tratamiento , Tirosina Quinasa c-MerRESUMEN
Optic nerve degeneration caused by glaucoma is a leading cause of blindness worldwide. Patients affected by the normal-pressure form of glaucoma are more likely to harbor risk alleles for glaucoma-related optic nerve disease. We have performed a meta-analysis of two independent genome-wide association studies for primary open angle glaucoma (POAG) followed by a normal-pressure glaucoma (NPG, defined by intraocular pressure (IOP) less than 22 mmHg) subgroup analysis. The single-nucleotide polymorphisms that showed the most significant associations were tested for association with a second form of glaucoma, exfoliation-syndrome glaucoma. The overall meta-analysis of the GLAUGEN and NEIGHBOR dataset results (3,146 cases and 3,487 controls) identified significant associations between two loci and POAG: the CDKN2BAS region on 9p21 (rs2157719 [G], ORâ=â0.69 [95%CI 0.63-0.75], pâ=â1.86×10⻹8), and the SIX1/SIX6 region on chromosome 14q23 (rs10483727 [A], ORâ=â1.32 [95%CI 1.21-1.43], pâ=â3.87×10⻹¹). In sub-group analysis two loci were significantly associated with NPG: 9p21 containing the CDKN2BAS gene (rs2157719 [G], ORâ=â0.58 [95% CI 0.50-0.67], pâ=â1.17×10⻹²) and a probable regulatory region on 8q22 (rs284489 [G], ORâ=â0.62 [95% CI 0.53-0.72], pâ=â8.88×10⻹°). Both NPG loci were also nominally associated with a second type of glaucoma, exfoliation syndrome glaucoma (rs2157719 [G], ORâ=â0.59 [95% CI 0.41-0.87], pâ=â0.004 and rs284489 [G], ORâ=â0.76 [95% CI 0.54-1.06], pâ=â0.021), suggesting that these loci might contribute more generally to optic nerve degeneration in glaucoma. Because both loci influence transforming growth factor beta (TGF-beta) signaling, we performed a genomic pathway analysis that showed an association between the TGF-beta pathway and NPG (permuted pâ=â0.009). These results suggest that neuro-protective therapies targeting TGF-beta signaling could be effective for multiple forms of glaucoma.
Asunto(s)
Síndrome de Exfoliación/genética , Estudio de Asociación del Genoma Completo , Glaucoma de Ángulo Abierto/genética , Degeneración Nerviosa , Factor de Crecimiento Transformador beta , Alelos , Cromosomas Humanos Par 8 , Cromosomas Humanos Par 9 , Proteínas de Homeodominio/genética , Humanos , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Nervio Óptico/patología , Polimorfismo de Nucleótido Simple , ARN Largo no Codificante , ARN no Traducido/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. Using genome-wide association single-nucleotide polymorphism data from the Glaucoma Genes and Environment study and National Eye Institute Glaucoma Human Genetics Collaboration comprising 3,108 cases and 3,430 controls, we assessed biologic pathways as annotated in the KEGG database for association with risk of POAG. After correction for genic overlap among pathways, we found 4 pathways, butanoate metabolism (hsa00650), hematopoietic cell lineage (hsa04640), lysine degradation (hsa00310) and basal transcription factors (hsa03022) related to POAG with permuted p < 0.001. In addition, the human leukocyte antigen (HLA) gene family was significantly associated with POAG (p < 0.001). In the POAG subset with normal-pressure glaucoma (NPG), the butanoate metabolism pathway was also significantly associated (p < 0.001) as well as the MAPK and Hedgehog signaling pathways (hsa04010 and hsa04340), glycosaminoglycan biosynthesis-heparan sulfate pathway (hsa00534) and the phenylalanine, tyrosine and tryptophan biosynthesis pathway (hsa0400). The butanoate metabolism pathway overall, and specifically the aspects of the pathway that contribute to GABA and acetyl-CoA metabolism, was the only pathway significantly associated with both POAG and NPG. Collectively these results implicate GABA and acetyl-CoA metabolism in glaucoma pathogenesis, and suggest new potential therapeutic targets.
Asunto(s)
Acetilcoenzima A/metabolismo , Glaucoma de Ángulo Abierto/genética , Glaucoma/genética , Redes y Vías Metabólicas/genética , Ácido gamma-Aminobutírico/metabolismo , Estudios de Casos y Controles , Análisis por Conglomerados , Femenino , Predisposición Genética a la Enfermedad , Glaucoma/metabolismo , Glaucoma de Ángulo Abierto/metabolismo , Humanos , Presión Intraocular/genética , Masculino , Modelos Genéticos , Polimorfismo de Nucleótido SimpleRESUMEN
PURPOSE: To evaluate the performance of N-[2-(diethylamino)ethyl]-(18)F-5-fluoropicolinamide ((18)F-P3BZA) for visualizing porcine retinal pigment epithelium (pRPE) cells transplanted in the striatum for the treatment of Parkinson disease and to monitor the long-term activity of implanted pRPE cells by means of (18)F-P3BZA positron emission tomography (PET)/computed tomography (CT) in vivo. MATERIALS AND METHODS: Animal work was conducted in accordance with the administrative panel on laboratory animal care. In vitro cell uptake of (18)F-P3BZA was determined with incubation of melanotic pRPE or amelanotic ARPE-19 cells with (18)F-P3BZA. To visualize the implanted pRPE cells in vivo, normal rats (four per group) were injected with pRPE or ARPE-19 cells attached to gelatin microcarriers in the left striatum and with control gelatin microcarriers in the right striatum and followed up with small animal PET/CT. Longitudinal PET/CT scans were acquired in 12 rats up to 16 days after surgery. Postmortem analysis, which included autoradiography and hematoxylin-eosin, Fontana-Masson, and immunofluorescence staining, was performed. Data were compared with the Student t test, analysis of variance, and regression analysis. RESULTS: (18)F-P3BZA accumulated in pRPE cells effectively (3.48% of the injected dose [ID] per gram of brain tissue ± 0.58 at 1 hour after injection of the probe at 2 days after surgery in vivo) but not in control ARPE-19 cells (P < .05). Longitudinal PET/CT scans revealed that the activity of implanted pRPE cells decreased over time, as evidenced by a reduction in (18)F-P3BZA uptake (3.39% ID/g ± 0.18, 2.49% ID/g ± 0.41, and 1.20% ID/g ± 0.13 at days 2, 9, and 16, respectively; P < .05). Postmortem analysis helped confirm the results of in vivo imaging. CONCLUSION: (18)F-P3BZA PET/CT is a feasible technique for visualizing and detecting the activity of implanted RPE cells in vivo.
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Radioisótopos de Flúor/farmacocinética , Imagen Molecular/métodos , Imagen Multimodal , Enfermedad de Parkinson/terapia , Ácidos Picolínicos/farmacocinética , Tomografía de Emisión de Positrones , Radiofármacos/farmacocinética , Epitelio Pigmentado de la Retina/trasplante , Tomografía Computarizada por Rayos X , Animales , Autorradiografía , Cuerpo Estriado , Femenino , Inmunohistoquímica , Ratas , Ratas Wistar , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/diagnóstico por imagen , Coloración y Etiquetado , PorcinosRESUMEN
PURPOSE: The CAV1/CAV2 (caveolin 1 and caveolin 2) genomic region previously was associated with primary open-angle glaucoma (POAG), although replication among independent studies has been variable. The aim of this study was to assess the association between CAV1/CAV2 single nucleotide polymorphisms (SNPs) and POAG in a large case-control dataset and to explore associations by gender and pattern of visual field (VF) loss further. DESIGN: Case-control study. PARTICIPANTS: We analyzed 2 large POAG data sets: the Glaucoma Genes and Environment (GLAUGEN) study (976 cases, 1140 controls) and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium (2132 cases, 2290 controls). METHODS: We studied the association between 70 SNPs located within the CAV1/CAV2 genomic region in the GLAUGEN and NEIGHBOR studies, both genotyped on the Illumina Human 660WQuadv1C BeadChip array and imputed with the Markov Chain Haplotyping algorithm using the HapMap 3 reference panel. We used logistic regression models of POAG in the overall population and separated by gender, as well as by POAG subtypes defined by type of VF defect (peripheral or paracentral). Results from GLAUGEN and NEIGHBOR were meta-analyzed, and a Bonferroni-corrected significance level of 7.7 × 10(-4) was used to account for multiple comparisons. MAIN OUTCOME MEASURES: Overall POAG, overall POAG by gender, and POAG subtypes defined by pattern of early VF loss. RESULTS: We found significant associations between 10 CAV1/CAV2 SNPs and POAG (top SNP, rs4236601; pooled P = 2.61 × 10(-7)). Of these, 9 were significant only in women (top SNP, rs4236601; pooled P = 1.59 × 10(-5)). Five of the 10 CAV1/CAV2 SNPs were associated with POAG with early paracentral VF (top SNP, rs17588172; pooled P = 1.07 × 10(-4)), and none of the 10 were associated with POAG with peripheral VF loss only or POAG among men. CONCLUSIONS: CAV1/CAV2 SNPs were associated significantly with POAG overall, particularly among women. Furthermore, we found an association between CAV1/CAV2 SNPs and POAG with paracentral VF defects. These data support a role for caveolin 1, caveolin 2, or both in POAG and suggest that the caveolins particularly may affect POAG pathogenesis in women and in patients with early paracentral VF defects.
Asunto(s)
Caveolina 1/genética , Caveolina 2/genética , Variación Estructural del Genoma , Glaucoma de Ángulo Abierto/genética , Polimorfismo de Nucleótido Simple , Trastornos de la Visión/genética , Campos Visuales , Anciano , Estudios de Casos y Controles , Femenino , Genotipo , Humanos , Presión Intraocular , Masculino , Persona de Mediana Edad , Factores SexualesRESUMEN
PURPOSE: Circulating estrogen levels are relevant in glaucoma phenotypic traits. We assessed the association between an estrogen metabolism single nucleotide polymorphism (SNP) panel in relation to primary open angle glaucoma (POAG), accounting for gender. METHODS: We included 3,108 POAG cases and 3,430 controls of both genders from the Glaucoma Genes and Environment (GLAUGEN) study and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium genotyped on the Illumina 660W-Quad platform. We assessed the relation between the SNP panels representative of estrogen metabolism and POAG using pathway- and gene-based approaches with the Pathway Analysis by Randomization Incorporating Structure (PARIS) software. PARIS executes a permutation algorithm to assess statistical significance relative to the pathways and genes of comparable genetic architecture. These analyses were performed using the meta-analyzed results from the GLAUGEN and NEIGHBOR data sets. We evaluated POAG overall as well as two subtypes of POAG defined as intraocular pressure (IOP) ≥22 mmHg (high-pressure glaucoma [HPG]) or IOP <22 mmHg (normal pressure glaucoma [NPG]) at diagnosis. We conducted these analyses for each gender separately and then jointly in men and women. RESULTS: Among women, the estrogen SNP pathway was associated with POAG overall (permuted p=0.006) and HPG (permuted p<0.001) but not NPG (permuted p=0.09). Interestingly, there was no relation between the estrogen SNP pathway and POAG when men were considered alone (permuted p>0.99). Among women, gene-based analyses revealed that the catechol-O-methyltransferase gene showed strong associations with HTG (permuted gene p≤0.001) and NPG (permuted gene p=0.01). CONCLUSIONS: The estrogen SNP pathway was associated with POAG among women.
Asunto(s)
Estrógenos/metabolismo , Predisposición Genética a la Enfermedad , Glaucoma de Ángulo Abierto/genética , Polimorfismo de Nucleótido Simple/genética , Caracteres Sexuales , Transducción de Señal/genética , Estudios de Casos y Controles , Femenino , Glaucoma de Ángulo Abierto/diagnóstico , Glaucoma de Ángulo Abierto/fisiopatología , Humanos , Presión Intraocular , Masculino , Redes y Vías Metabólicas/genética , Estados UnidosRESUMEN
Primary cilia are conserved organelles that integrate extracellular cues into intracellular signals and are critical for diverse processes, including cellular development and repair responses. Deficits in ciliary function cause multisystemic human diseases known as ciliopathies. In the eye, atrophy of the retinal pigment epithelium (RPE) is a common feature of many ciliopathies. However, the roles of RPE cilia in vivo remain poorly understood. In this study, we first found that mouse RPE cells only transiently form primary cilia. We then examined the RPE in the mouse model of Bardet-Biedl Syndrome 4 (BBS4), a ciliopathy associated with retinal degeneration in humans, and found that ciliation in BBS4 mutant RPE cells is disrupted early during development. Next, using a laser-induced injury model in vivo, we found that primary cilia in RPE reassemble in response to laser injury during RPE wound healing and then rapidly disassemble after the repair is completed. Finally, we demonstrated that RPE-specific depletion of primary cilia in a conditional mouse model of cilia loss promoted wound healing and enhanced cell proliferation. In summary, our data suggest that RPE cilia contribute to both retinal development and repair and provide insights into potential therapeutic targets for more common RPE degenerative diseases.
Asunto(s)
Ciliopatías , Degeneración Retiniana , Ratones , Humanos , Animales , Epitelio Pigmentado de la Retina , Cilios/fisiología , Modelos Animales de Enfermedad , Proteínas Supresoras de Tumor , Proteínas Asociadas a MicrotúbulosRESUMEN
Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer's disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/ß-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD4-specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.
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Barrera Hematoencefálica , Receptores Frizzled , Ratones , Animales , Barrera Hematoencefálica/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Retina/metabolismo , Barrera Hematorretinal/metabolismo , Vía de Señalización WntRESUMEN
PURPOSE: In a previous study, several quantitative trait loci (QTL) that influence age-related degeneration (ageRD) were identified in a cross between the albino strains B6(Cg)-Tyr(c-2J)/J (B6a) and BALB/cByJ (C). The Chromosome (Chr) 6 and Chr 10 QTL were the strongest and most highly significant loci and both involved B6a protective alleles. The QTL were responsible for 21% and 9% of the variance in phenotypes, respectively. We focused on these two QTL to identify candidate genes. METHODS: DNA microarrays were used for the two mouse strains at four and eight months of age to identify genes that are differentially regulated and map to either QTL. Gene Ontology (GO) analysis of the differentially expressed genes was performed to identify possible processes and pathways associated with ageRD. To identify additional candidates, database analyses (Positional Medline or PosMed) were used. Based on differential expression, PosMed, and the presence of reported polymorphisms, five genes per QTL were selected for further study by sequencing analysis and qRT-PCR. Tumor necrosis factor, alpha- induced protein 3 (Tnfaip3; on a C57BL/6J (B6) background) was phenotypically tested. Single nucleotide polymorphisms (SNPs) flanking this gene were correlated with outer nuclear layer thickness (ONL), and eight-month-old Tnfaip3(+/-) mice were tested for ageRD. RESULTS: Polymorphisms were found in the coding regions of eight genes. Changes in gene expression were identified by qRT-PCR for Hexokinase 2 (Hk2) and Docking protein 1 (Dok1) at four months and for Dok1 and Tnfaip3 at eight months. Tnfaip3 was selected for phenotypic testing due to differential expression and the presence of two nonsynonymous mutations. However, when ONL thickness was compared in eight-month-old congenic Tnfaip3(+/-) and Tnfaip3(+/+) mice, no differences were found, suggesting that Tnfaip3 is not the quantitative trait gene (QTG) for the Chr 10 QTL. The GO analysis revealed that GO terms associated with stress and cell remodeling are overrepresented in the ageRD-sensitive C strain compared with the B6a strain with age (eight months). In the ageRD-resistant B6a strain, compared with the C strain, GO terms associated with antioxidant response and the regulation of blood vessel size are overrepresented with age. CONCLUSIONS: The analyses of differentially expressed genes and the PosMed database yielded candidate genes for the Chr 6 and Chr 10 QTL. HtrA serine peptidase 2 (Htra2), Dok1, and Tnfaip3 were deemed most promising because of their known roles in apoptosis and our finding of nonsynonymous substitutions between B6a and C strains. While Tnfaip3 was excluded as the QTG for the Chr 10 QTL, Dok1 and Htra2 remain good candidates for the Chr 6 QTL. Finally, the GO term analysis further supports the general hypothesis that oxidative stress is involved in ageRD.
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Mapeo Cromosómico , Estudios de Asociación Genética , Sitios de Carácter Cuantitativo , Degeneración Retiniana/genética , Animales , Cisteína Endopeptidasas/genética , Proteínas de Unión al ADN/genética , Femenino , Serina Peptidasa A2 que Requiere Temperaturas Altas , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos , Ratones Noqueados , Análisis por Micromatrices , Proteínas Mitocondriales/genética , Estrés Oxidativo , Fenotipo , Fosfoproteínas/genética , Polimorfismo de Nucleótido Simple , Proteínas de Unión al ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
Mitochondrial diseases are a clinically heterogenous group of disorders caused by respiratory chain dysfunction and associated with progressive, multi-systemic phenotype. There is no effective treatment or cure, and no FDA-approved drug for treating mitochondrial disease. To identify and characterize potential therapeutic compounds, we developed an in vitro screening assay and identified a group of direct AMP-activated protein kinase (AMPK) activators originally developed for the treatment of diabetes and metabolic syndrome. Unlike previously investigated AMPK agonists such as AICAR, these compounds allosterically activate AMPK in an AMP-independent manner, thereby increasing specificity and decreasing pleiotropic effects. The direct AMPK activator PT1 significantly improved mitochondrial function in assays of cellular respiration, energy status, and cellular redox. PT1 also protected against retinal degeneration in a mouse model of photoreceptor degeneration associated with mitochondrial dysfunction and oxidative stress, further supporting the therapeutic potential of AMP-independent AMPK agonists in the treatment of mitochondrial disease.
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Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Enfermedades Mitocondriales/tratamiento farmacológico , Tiazoles/administración & dosificación , metaminobenzoatos/administración & dosificación , Regulación Alostérica/efectos de los fármacos , Animales , Compuestos de Bifenilo , Respiración de la Célula/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Ratones , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Estrés Oxidativo/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Pironas/administración & dosificación , Pironas/farmacología , Tiazoles/farmacología , Tiofenos/administración & dosificación , Tiofenos/farmacología , metaminobenzoatos/farmacologíaRESUMEN
We investigated the effects of treating differentiated retinal pigment epithelial (RPE) cells with didanosine (ddI), which is associated with retinopathy in individuals with HIV/AIDS. We hypothesized that such treatment would cause depletion of mitochondrial DNA and provide insight into the consequences of degradation of RPE mitochondrial function in aging and disease. Treatment of differentiated ARPE-19 or human primary RPE cells with 200 µM ddI for 6-24 days was not cytotoxic but caused up to 60% depletion of mitochondrial DNA, and a similar reduction in mitochondrial membrane potential and NDUFA9 protein abundance. Mitochondrial DNA-depleted RPE cells demonstrated enhanced aerobic glycolysis by extracellular flux analysis, increased AMP kinase activation, reduced mTOR activity, and increased resistance to cell death in response to treatment with the oxidant, sodium iodate. We conclude that ddI-mediated mitochondrial DNA depletion promotes a glycolytic shift in differentiated RPE cells and enhances resistance to oxidative damage. Our use of ddI treatment to induce progressive depletion of mitochondrial DNA in differentiated human RPE cells should be widely applicable for other studies aimed at understanding RPE mitochondrial dysfunction in aging and disease.
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Diferenciación Celular/genética , ADN Mitocondrial/metabolismo , Células Epiteliales/metabolismo , Epitelio Pigmentado de la Retina/citología , Adenilato Quinasa/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Didanosina/farmacología , Células Epiteliales/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
Purpose: The accumulation of undigestible autofluorescent material (UAM), termed lipofuscin in vivo, is a hallmark of aged RPE. Lipofuscin derives, in part, from the incomplete degradation of phagocytized photoreceptor outer segments (OS). Whether this accumulated waste is toxic is unclear. We therefore investigated the effects of UAM in highly differentiated human fetal RPE (hfRPE) cultures. Methods: Unmodified and photo-oxidized OS were fed daily to confluent cultures of ARPE-19 RPE or hfRPE. The emission spectrum, composition, and morphology of resulting UAM were measured and compared to in vivo lipofuscin. Effects of UAM on multiple RPE phenotypes were assessed. Results: Compared to ARPE-19, hfRPE were markedly less susceptible to UAM buildup. Accumulated UAM in hfRPE initially resembled the morphology of lipofuscin from AMD eyes, but compacted and shifted spectrum over time to resemble lipofuscin from healthy aged human RPE. UAM accumulation mildly reduced transepithelial electrical resistance, ketogenesis, certain RPE differentiation markers, and phagocytosis efficiency, while inducing senescence and rare, focal pockets of epithelial-mesenchymal transition. However, it had no effects on mitochondrial oxygen consumption rate, certain other RPE differentiation markers, secretion of drusen components or polarity markers, nor cell death. Conclusions: hfRPE demonstrates a remarkable resistance to UAM accumulation, suggesting mechanisms for efficient OS processing that may be lost in other RPE culture models. Furthermore, while UAM alters hfRPE phenotype, the effects are modest, consistent with conflicting reports in the literature on the toxicity of lipofuscin. Our results suggest that healthy RPE may adequately adapt to and tolerate lipofuscin accumulation.