RESUMEN
Rhodopsin misfolding caused by the P23H mutation is a major cause of autosomal dominant retinitis pigmentosa (adRP). To date, there are no effective treatments for adRP. The BiP co-chaperone and reductase ERdj5 (DNAJC10) is part of the endoplasmic reticulum (ER) quality control machinery, and previous studies have shown that overexpression of ERdj5 in vitro enhanced the degradation of P23H rhodopsin, whereas knockdown of ERdj5 increased P23H rhodopsin ER retention and aggregation. Here, we investigated the role of ERdj5 in photoreceptor homeostasis in vivo by using an Erdj5 knockout mouse crossed with the P23H knock-in mouse and by adeno-associated viral (AAV) vector-mediated gene augmentation of ERdj5 in P23H-3 rats. Electroretinogram (ERG) and optical coherence tomography of Erdj5-/- and P23H+/-:Erdj5-/- mice showed no effect of ERdj5 ablation on retinal function or photoreceptor survival. Rhodopsin levels and localization were similar to those of control animals at a range of time points. By contrast, when AAV2/8-ERdj5-HA was subretinally injected into P23H-3 rats, analysis of the full-field ERG suggested that overexpression of ERdj5 reduced visual function loss 10 weeks post-injection (PI). This correlated with a significant preservation of photoreceptor cells at 4 and 10 weeks PI. Assessment of the outer nuclear layer (ONL) morphology showed preserved ONL thickness and reduced rhodopsin retention in the ONL in the injected superior retina. Overall, these data suggest that manipulation of the ER quality control and ER-associated degradation factors to promote mutant protein degradation could be beneficial for the treatment of adRP caused by mutant rhodopsin.
Asunto(s)
Proteínas del Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Retinitis Pigmentosa/genética , Rodopsina/genética , Animales , Modelos Animales de Enfermedad , Electrorretinografía , Retículo Endoplásmico/genética , Técnicas de Sustitución del Gen , Ratones , Ratones Noqueados , Mutación/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Ratas , Retina/metabolismo , Retina/patología , Retinitis Pigmentosa/patología , Rodopsina/metabolismo , TransfecciónRESUMEN
The retinal vasculature is tightly organized in a structure that provides for the high metabolic demand of neurons while minimizing interference with incident light. The adverse impact of retinal vascular insufficiency is mitigated by adaptive vascular regeneration but exacerbated by pathological neovascularization. Aberrant growth of neovessels in the retina is responsible for impairment of sight in common blinding disorders including retinopathy of prematurity, proliferative diabetic retinopathy, and age-related macular degeneration. Myeloid cells are key players in this process, with diverse roles that can either promote or protect against ocular neovascularization. We have previously demonstrated that myeloid-derived VEGF, HIF1, and HIF2 are not essential for pathological retinal neovascularization. Here, however, we show by cell-specific depletion of Vhl in a mouse model of retinal ischemia (oxygen-induced retinopathy, OIR) that myeloid-derived HIFs promote VEGF and bFGF expression and enhance vascular regeneration in association with improved density and organization of the astrocytic network.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Isquemia/genética , Células Mieloides/metabolismo , Regeneración/genética , Vasos Retinianos/fisiología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula/genética , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Modelos Animales de Enfermedad , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isquemia/metabolismo , Isquemia/patología , Ratones , Ratones Transgénicos , Retina/patología , Enfermedades de la Retina/genética , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
In the adult central nervous system, endothelial and neuronal cells engage in tight cross-talk as key components of the so-called neurovascular unit. Impairment of this important relationship adversely affects tissue homeostasis, as observed in neurodegenerative conditions including Alzheimer's and Parkinson's disease. In development, the influence of neuroprogenitor cells on angiogenesis is poorly understood. Here, we show in mouse that these cells interact intimately with the growing retinal vascular network, and we identify a novel regulatory mechanism of vasculature development mediated by hypoxia-inducible factor 2a (Hif2a). By Cre-lox gene excision, we show that Hif2a in retinal neuroprogenitor cells upregulates the expression of the pro-angiogenic mediators vascular endothelial growth factor and erythropoietin, whereas it locally downregulates the angiogenesis inhibitor endostatin. Importantly, absence of Hif2a in retinal neuroprogenitor cells causes a marked reduction of proliferating endothelial cells at the angiogenic front. This results in delayed retinal vascular development, fewer major retinal vessels and reduced density of the peripheral deep retinal vascular plexus. Our findings demonstrate that retinal neuroprogenitor cells are a crucial component of the developing neurovascular unit.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Vasos Retinianos/crecimiento & desarrollo , Vasos Retinianos/inervación , Animales , Astrocitos/citología , Astrocitos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proliferación Celular , Endostatinas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neovascularización Fisiológica/genética , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Epitelio Pigmentado de la Retina/crecimiento & desarrollo , Epitelio Pigmentado de la Retina/metabolismo , Vasos Retinianos/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage disorders characterized by general neurodegeneration and premature death. Sight loss is also a major symptom in NCLs, severely affecting the quality of life of patients, but it is not targeted effectively by brain-directed therapies. Here we set out to explore the therapeutic potential of an ocular gene therapy to treat sight loss in NCL due to a deficiency in the transmembrane protein CLN6. We found that, although Cln6nclf mice presented mainly with photoreceptor degeneration, supplementation of CLN6 in photoreceptors was not beneficial. Because the level of CLN6 is low in photoreceptors but high in bipolar cells (retinal interneurons that are only lost in Cln6-deficient mice at late disease stages), we explored the therapeutic effects of delivering CLN6 to bipolar cells using adeno-associated virus (AAV) serotype 7m8. Bipolar cell-specific expression of CLN6 slowed significantly the loss of photoreceptor function and photoreceptor cells. This study shows that the deficiency of a gene normally expressed in bipolar cells can cause the loss of photoreceptors and that this can be prevented by bipolar cell-directed treatment.
Asunto(s)
Proteínas de la Membrana/genética , Lipofuscinosis Ceroideas Neuronales/genética , Células Fotorreceptoras/metabolismo , Células Bipolares de la Retina/metabolismo , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/genética , Humanos , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Lipofuscinosis Ceroideas Neuronales/metabolismo , Lipofuscinosis Ceroideas Neuronales/patología , Lipofuscinosis Ceroideas Neuronales/terapia , Células Fotorreceptoras/patologíaRESUMEN
Human vision relies heavily upon cone photoreceptors, and their loss results in permanent visual impairment. Transplantation of healthy photoreceptors can restore visual function in models of inherited blindness, a process previously understood to arise by donor cell integration within the host retina. However, we and others recently demonstrated that donor rod photoreceptors engage in material transfer with host photoreceptors, leading to the host cells acquiring proteins otherwise expressed only by donor cells. We sought to determine whether stem cell- and donor-derived cones undergo integration and/or material transfer. We find that material transfer accounts for a significant proportion of rescued cells following cone transplantation into non-degenerative hosts. Strikingly, however, substantial numbers of cones integrated into the Nrl-/- and Prph2rd2/rd2, but not Nrl-/-;RPE65R91W/R91W, murine models of retinal degeneration. This confirms the occurrence of photoreceptor integration in certain models of retinal degeneration and demonstrates the importance of the host environment in determining transplantation outcome.
Asunto(s)
Ceguera/terapia , Células Fotorreceptoras Retinianas Conos/trasplante , Degeneración Retiniana/terapia , Trasplante de Células Madre , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Ceguera/genética , Ceguera/patología , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Humanos , Ratones , Periferinas/genética , Retina/patología , Retina/trasplante , Células Fotorreceptoras Retinianas Conos/citología , Degeneración Retiniana/patología , Células Madre/citología , cis-trans-Isomerasas/genéticaRESUMEN
Transplantation of rod photoreceptors, derived either from neonatal retinae or pluripotent stem cells (PSCs), can restore rod-mediated visual function in murine models of inherited blindness. However, humans depend more upon cone photoreceptors that are required for daylight, color, and high-acuity vision. Indeed, macular retinopathies involving loss of cones are leading causes of blindness. An essential step for developing stem cell-based therapies for maculopathies is the ability to generate transplantable human cones from renewable sources. Here, we report a modified 2D/3D protocol for generating hPSC-derived neural retinal vesicles with well-formed ONL-like structures containing cones and rods bearing inner segments and connecting cilia, nascent outer segments, and presynaptic structures. This differentiation system recapitulates human photoreceptor development, allowing the isolation and transplantation of a pure population of stage-matched cones. Purified human long/medium cones survive and become incorporated within the adult mouse retina, supporting the potential of photoreceptor transplantation for treating retinal degeneration.
Asunto(s)
Células Madre Pluripotentes/citología , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Conos/trasplante , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/ultraestructura , Humanos , Células Madre Pluripotentes/metabolismo , Degeneración Retiniana/patología , Degeneración Retiniana/terapia , Factores de TiempoRESUMEN
Retinal ischemia and pathological angiogenesis cause severe impairment of sight. Oxygen-induced retinopathy (OIR) in young mice is widely used as a model to investigate the underlying pathological mechanisms and develop therapeutic interventions. We compared directly the conventional OIR model (exposure to 75% O2 from postnatal day (P) 7 to P12) with an alternative, accelerated version (85% O2 from P8 to P11). We found that accelerated OIR induces similar pre-retinal neovascularization but greater retinal vascular regression that recovers more rapidly. The extent of retinal gliosis is similar but neuroretinal function, as measured by electroretinography, is better maintained in the accelerated model. We found no systemic or maternal morbidity in either model. Accelerated OIR offers a safe, reliable and more rapid alternative model in which pre-retinal neovascularization is similar but retinal vascular regression is greater.
Asunto(s)
Neovascularización Retiniana/etiología , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Humanos , Hiperoxia/complicaciones , Isquemia/complicaciones , Isquemia/patología , Isquemia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Regeneración , Enfermedades de la Retina/etiología , Enfermedades de la Retina/patología , Enfermedades de la Retina/fisiopatología , Neovascularización Retiniana/patología , Neovascularización Retiniana/fisiopatología , Vasos Retinianos/patología , Vasos Retinianos/fisiopatología , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor ß2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1-/- mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.
Asunto(s)
Células Madre Embrionarias de Ratones/trasplante , Células Fotorreceptoras Retinianas Conos/citología , Degeneración Retiniana/terapia , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/antagonistas & inhibidores , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Proteínas del Ojo/antagonistas & inhibidores , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Factor Nuclear 6 del Hepatocito/metabolismo , Factor Inhibidor de Leucemia/farmacología , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/citología , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Opsinas/metabolismo , Receptores Nucleares Huérfanos/antagonistas & inhibidores , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/metabolismo , Factores de Transcripción Otx/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Notch/antagonistas & inhibidores , Receptores Notch/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Degeneración Retiniana/patología , Transducción de Señal , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
The impact of many inflammatory diseases is influenced by age-related changes in the activation of resident and circulating myeloid cells. In the eye, a major sight-threatening consequence of age-related macular degeneration is the development of severe choroidal neovascularization (CNV). To identify the molecular pathways and myeloid cell populations involved in this increased neovascular response, we characterized the immune status of murine choroid and retina during aging and in the context of experimental CNV. In the choroid, but not in the retina, advancing age is associated with proinflammatory upregulation of CCL2-CCR2 signaling. Genetic excision of CCL2 diminishes age-related inflammatory changes in the choroid, with reduced recruitment of proinflammatory myeloid cells and attenuation of CNV. These findings indicate that CCL2-driven recruitment of myeloid cells contributes to increased severity of CNV with age. Similar mechanisms may be involved in other age-related inflammatory diseases.
Asunto(s)
Envejecimiento/genética , Envejecimiento/patología , Quimiocina CCL2/fisiología , Neovascularización Coroidal/genética , Neovascularización Coroidal/patología , Receptores CCR2/fisiología , Transducción de Señal , Animales , Células Cultivadas , Ratones Endogámicos C57BL , Ratones Noqueados , Índice de Severidad de la EnfermedadRESUMEN
Dysregulation of the complement system has been implicated in the pathogenesis of age-related macular degeneration. To investigate consequences of altered complement regulation in the eye with age, we examined Cd59a complement regulator deficient (Cd59a(-/-)) mice between 4 and 15 months. In vivo imaging revealed an increased age-related accumulation of autofluorescent spots in Cd59a(-/-) mice, a feature that reflects accumulation of subretinal macrophages and/or microglia. Despite this activation of myeloid cells in the eye, Cd59a(-/-) mice showed normal retinal histology and function as well as normal choroidal microvasculature. With age, they revealed increased expression of activators of the alternative complement pathway (C3, Cfb, Cfd), in particular in the retinal pigment epithelium (RPE)-choroid but less in the retina. This molecular response was not altered by moderately-enhanced light exposure. Cd59a deficiency therefore leads to a preferential age-related dysregulation of the complement system in the RPE-choroid, that alone or in combination with light as a trigger, is not sufficient to cause choroidal vascular changes or retinal degeneration and dysfunction. This data emphasizes the particular vulnerability of the RPE-choroidal complex to dysregulation of the alternative complement pathway during aging.
Asunto(s)
Envejecimiento/genética , Antígenos CD59/metabolismo , Coroides/metabolismo , Factores Inmunológicos/metabolismo , Degeneración Macular , Epitelio Pigmentado de la Retina/metabolismo , Análisis de Varianza , Animales , Antígenos CD59/genética , Coroides/patología , Activación de Complemento , Modelos Animales de Enfermedad , Electrorretinografía , Regulación de la Expresión Génica/genética , Macrófagos/metabolismo , Macrófagos/patología , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Microglía/patología , Proteínas del Tejido Nervioso/genética , ARN Mensajero/metabolismo , Retina/patología , Epitelio Pigmentado de la Retina/patologíaRESUMEN
The rd1 mouse with a mutation in the Pde6b gene was the first strain of mice identified with a retinal degeneration. However, AAV-mediated gene supplementation of rd1 mice only results in structural preservation of photoreceptors, and restoration of the photoreceptor-mediated a-wave, but not in restoration of the bipolar cell-mediated b-wave. Here we show that a mutation in Gpr179 prevents the full restoration of vision in rd1 mice. Backcrossing rd1 with C57BL6 mice reveals the complete lack of b-wave in a subset of mice, consistent with an autosomal recessive Mendelian inheritance pattern. We identify a mutation in the Gpr179 gene, which encodes for a G-protein coupled receptor localized to the dendrites of ON-bipolar cells. Gene replacement in rd1 mice that are devoid of the mutation in Gpr179 successfully restores the function of both photoreceptors and bipolar cells, which is maintained for up to 13 months. Our discovery may explain the failure of previous gene therapy attempts in rd1 mice, and we propose that Grp179 mutation status should be taken into account in future studies involving rd1 mice.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Terapia Genética/métodos , Receptores Acoplados a Proteínas G/genética , Degeneración Retiniana/genética , Animales , Cruzamientos Genéticos , Dependovirus , Electrorretinografía/métodos , Miedo , Femenino , Fondo de Ojo , Genotipo , Homocigoto , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Genéticos , Mutación , Plásmidos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Degeneración Retiniana/metabolismo , Factores de Tiempo , Tomografía de Coherencia ÓpticaRESUMEN
Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair and restoration of vision through transplantation of photoreceptor precursors obtained from postnatal retinas into visually impaired adult mice. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs. We show that rod precursors derived by this protocol and selected via a GFP reporter under the control of a Rhodopsin promoter integrate within degenerate retinas of adult mice and mature into outer segment-bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently compared with cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.
Asunto(s)
Ceguera/terapia , Técnicas de Cultivo de Célula , Células Madre Embrionarias/trasplante , Retina/citología , Animales , Ceguera/genética , Ceguera/patología , Diferenciación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Células Madre Embrionarias/citología , Proteínas Fluorescentes Verdes , Ratones , Células Fotorreceptoras , Células Fotorreceptoras de Vertebrados , Retina/metabolismo , Retina/patología , Rodopsina/genética , Rodopsina/metabolismo , Trasplante de Células MadreRESUMEN
Monocytes, macrophages, dendritic cells and microglia play critical roles in the local immune response to acute and chronic tissue injury and have been implicated in the pathogenesis of age-related macular degeneration. Defects in Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling cause enhanced accumulation of bloated subretinal microglia/macrophages in senescent mice and this phenomenon is reported to result in the acceleration of age-related retinal degeneration. The purpose of this study was to determine whether defects in CCL2-CCR2 and CX3CL1-CX3CR1 signalling pathways, alone or in combination, cause age-dependent retinal degeneration. We tested whether three chemokine knockout mouse lines, Ccl2(-/-), Cx3cr1(-/-) and Ccl2(-/-)/Cx3cr1(-/-), in comparison to age-matched C57Bl/6 control mice show differences in subretinal macrophage accumulation and loss of adjacent photoreceptor cells at 12-14 months of age. All mouse lines are derived from common parental strains and do not carry the homozygous rd8 mutation in the Crb1 gene that has been a major confounding factor in previous reports. We quantified subretinal macrophages by counting autofluorescent lesions in fundus images obtained by scanning laser ophthalmoscopy (AF-SLO) and by immunohistochemistry for Iba1 positive cells. The accumulation of subretinal macrophages was enhanced in Ccl2(-/-), but not in Cx3cr1(-/-) or Ccl2(-/-)/Cx3cr1(-/-) mice. We identified no evidence of retinal degeneration in any of these mouse lines by TUNEL staining or semithin histology. In conclusion, CCL2-CCR2 and/or CX3CL1-CX3CR1 signalling defects may differentially affect the trafficking of microglia and macrophages in the retina during ageing, but do not appear to cause age-related retinal degeneration in mice.
Asunto(s)
Quimiocina CCL2/fisiología , Degeneración Macular/metabolismo , Receptores de Quimiocina/fisiología , Animales , Receptor 1 de Quimiocinas CX3C , Proteínas de Unión al Calcio/metabolismo , Recuento de Células , Genotipo , Etiquetado Corte-Fin in Situ , Macrófagos/metabolismo , Degeneración Macular/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Oftalmoscopía , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Reacción en Cadena de la PolimerasaRESUMEN
Despite different aetiologies, age-related macular degeneration and most inherited retinal disorders culminate in the same final common pathway, the loss of photoreceptors. There are few treatments and none reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy applicable to all degenerations. Recently, we demonstrated restoration of vision following rod-photoreceptor transplantation into a mouse model of stationary night-blindness, raising the critical question of whether photoreceptor replacement is equally effective in different types and stages of degeneration. We present a comprehensive assessment of rod-photoreceptor transplantation across six murine models of inherited photoreceptor degeneration. Transplantation is feasible in all models examined but disease type has a major impact on outcome, as assessed both by the morphology and number of integrated rod-photoreceptors. Integration can increase (Prph2(+/Δ307)), decrease (Crb1(rd8/rd8), Gnat1(-/-), Rho(-/-)), or remain constant (PDE6ß(rd1/rd1), Prph2(rd2/rd2)) with disease progression, depending upon the gene defect, with no correlation with severity. Robust integration is possible even in late-stage disease. Glial scarring and outer limiting membrane integrity, features that change with degeneration, significantly affect transplanted photoreceptor integration. Combined breakdown of these barriers markedly increases integration in a model with an intact outer limiting membrane, strong gliotic response, and otherwise poor transplantation outcome (Rho(-/-)), leading to an eightfold increase in integration and restoration of visual function. Thus, it is possible to achieve robust integration across a broad range of inherited retinopathies. Moreover, transplantation outcome can be improved by administering appropriate, tailored manipulations of the recipient environment.
Asunto(s)
Ceguera Nocturna/cirugía , Atrofia Óptica Hereditaria de Leber/cirugía , Células Fotorreceptoras Retinianas Bastones/trasplante , Retinitis Pigmentosa/cirugía , Animales , Western Blotting , Recuento de Células , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Citometría de Flujo , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica de Transmisión , Proteínas del Tejido Nervioso/metabolismo , Ceguera Nocturna/genética , Atrofia Óptica Hereditaria de Leber/genética , Periferinas , Células Fotorreceptoras Retinianas Bastones/ultraestructura , Retinitis Pigmentosa/genética , Transducina/genética , Transducina/metabolismo , Resultado del Tratamiento , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Because retinal ischemia is a common cause of vision loss, we sought to determine the effects of ischemia on neuroretinal function and survival in murine oxygen-induced retinopathy (OIR) and to define the role of endogenous erythropoietin (EPO) in this model. OIR is a reproducible model of ischemia-induced retinal neovascularization; it is used commonly to develop antiangiogenic strategies. We investigated the effects of ischemia in murine OIR on retinal function and neurodegeneration by electroretinography and detailed morphology. OIR was associated with significant neuroretinal dysfunction, with reduced photopic and scotopic ERG responses and reduced b-wave/a-wave ratios consistent with specific inner-retinal dysfunction. OIR resulted in significantly increased apoptosis and atrophy of the inner retina in areas of ischemia. EPO deficiency in heterozygous Epo-Tag transgenic mice was associated with more profound retinal dysfunction after OIR, indicated by a significantly greater suppression of ERG amplitudes, but had no measurable effect on the extent of retinal ischemia, preretinal neovascularization, or neuroretinal degeneration in OIR. Systemic administration of recombinant EPO protected EPO-deficient mice against this additional suppression, but EPO supplementation in wild-type animals with OIR did not rescue neuroretinal dysfunction or degeneration. Murine OIR offers a valuable model of ischemic neuroretinal dysfunction and degeneration in which to investigate adaptive tissue responses and evaluate novel therapeutic approaches. Endogenous EPO can protect neuroretinal function in ischemic retinopathy.
Asunto(s)
Eritropoyetina/fisiología , Isquemia/fisiopatología , Neuronas Retinianas/fisiología , Vasos Retinianos/patología , Animales , Apoptosis/fisiología , Hipoxia de la Célula/fisiología , Modelos Animales de Enfermedad , Electrorretinografía/métodos , Eritropoyetina/biosíntesis , Eritropoyetina/deficiencia , Eritropoyetina/uso terapéutico , Isquemia/complicaciones , Riñón/metabolismo , Ratones , Ratones Transgénicos , Oxígeno , Proteínas Recombinantes/uso terapéutico , Retina/metabolismo , Retina/patología , Neovascularización Retiniana/etiología , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Neuronas Retinianas/patología , Regulación hacia ArribaRESUMEN
Cone dystrophy 3 (COD3) is a severe dominantly inherited retinal degeneration caused by missense mutations in GUCA1A, the gene encoding Guanylate Cyclase Activating Protein 1 (GCAP1). The role of GCAP1 in controlling cyclic nucleotide levels in photoreceptors has largely been elucidated using knock-out mice, but the disease pathology in these mice cannot be extrapolated directly to COD3 as this involves altered, rather than loss of, GCAP1 function. Therefore, in order to evaluate the pathology of this dominant disorder, we have introduced a point mutation into the murine Guca1a gene that causes an E155G amino acid substitution; this is one of the disease-causing mutations found in COD3 patients. Disease progression in this novel mouse model of cone dystrophy was determined by a variety of techniques including electroretinography (ERG), retinal histology, immunohistochemistry and measurement of cGMP levels. It was established that although retinal development was normal up to 3 months of age, there was a subsequent progressive decline in retinal function, with a far greater alteration in cone than rod responses, associated with a corresponding loss of photoreceptors. In addition, we have demonstrated that accumulation of cyclic GMP precedes the observed retinal degeneration and is likely to contribute to the disease mechanism. Importantly, this knock-in mutant mouse has many features in common with the human disease, thereby making it an excellent model to further probe disease pathogenesis and investigate therapeutic interventions.
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Marcación de Gen , Genes Dominantes/genética , Proteínas Activadoras de la Guanilato-Ciclasa/genética , Retinitis Pigmentosa/genética , Animales , Enfermedades Asintomáticas , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Técnicas de Sustitución del Gen , Proteínas Activadoras de la Guanilato-Ciclasa/metabolismo , Humanos , Espacio Intracelular/metabolismo , Cinética , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Retinitis Pigmentosa/fisiopatologíaRESUMEN
Stem cell therapy presents an opportunity to replace photoreceptors that are lost as a result of inherited and age-related degenerative disease. We have previously shown that murine postmitotic rod photoreceptor precursor cells, identified by expression of the rod-specific transcription factor Nrl, are able to migrate into and integrate within the adult murine neural retina. However, their long-term survival has yet to be determined. Here, we found that integrated Nrl.gfp(+ve) photoreceptors were present up to 12 months post-transplantation, albeit in significantly reduced numbers. Surviving cells had rod-like morphology, including inner/outer segments and spherule synapses. In a minority of eyes, we observed an early, marked reduction in integrated photoreceptors within 1 month post-transplantation, which correlated with increased numbers of amoeboid macrophages, indicating acute loss of transplanted cells due to an inflammatory response. In the majority of transplants, similar numbers of integrated cells were observed between 1 and 2 months post-transplantation. By 4 months, however, we observed a significant decrease in integrated cell survival. Macrophages and T cells were present around the transplantation site, indicating a chronic immune response. Immune suppression of recipients significantly increased transplanted photoreceptor survival, indicating that the loss observed in unsuppressed recipients resulted from T cell-mediated host immune responses. Thus, if immune responses are modulated, correctly integrated transplanted photoreceptors can survive for extended periods of time in hosts with partially mismatched H-2 haplotypes. These findings suggest that autologous donor cells are optimal for therapeutic approaches to repair the neural retina, though with immune suppression nonautologous donors may be effective.
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Células Fotorreceptoras/citología , Retina/citología , Trasplante de Células Madre/métodos , Animales , Supervivencia Celular/inmunología , Células Cultivadas , Ciclosporina/uso terapéutico , Citometría de Flujo , Inmunohistoquímica , Inmunosupresores/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Células Fotorreceptoras/inmunología , Células Fotorreceptoras/metabolismo , Retina/efectos de los fármacos , Retina/inmunología , Retina/metabolismo , Linfocitos T/inmunología , Factores de TiempoRESUMEN
BACKGROUND: Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO. CONCLUSIONS/SIGNIFICANCE: Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isquemia/metabolismo , Vasos Retinianos/metabolismo , Animales , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Eritropoyetina/metabolismo , Inmunohistoquímica , Ratones , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PURPOSE: Drusen, which are defined clinically as yellowish white spots in the outer retina, are cardinal features of age-related macular degeneration (AMD). Ccl2-knockout (Ccl2(-/-)) mice have been reported to develop drusen and phenotypic features similar to AMD, including an increased susceptibility to choroidal neovascularization (CNV). This study was conducted to investigate the nature of the drusenlike lesions in vivo and further evaluate the Ccl2(-/-) mouse as a model of AMD. METHODS: The eyes of 2- to 25-month-old Ccl2(-/-) and C57Bl/6 mice were examined in vivo by autofluorescence scanning laser ophthalmoscopy (AF-SLO) and electroretinography, and the extent of laser-induced CNV was measured by fluorescein fundus angiography. The retinal morphology was also assessed by immunohistochemistry and quantitative histologic and ultrastructural morphometry. RESULTS: The drusenlike lesions of Ccl2(-/-) mice comprised accelerated accumulation of swollen CD68(+), F4/80(+) macrophages in the subretinal space that were apparent as autofluorescent foci on AF-SLO. These macrophages contained pigment granules and phagosomes with outer segment and lipofuscin inclusions that may account for their autofluorescence. Only age-related retinal pigment epithelium (RPE) damage, photoreceptor loss, and sub-RPE deposits were observed but, despite the accelerated accumulation of macrophages, we identified no spontaneous development of CNV in the senescent mice and found a reduced susceptibility to laser-induced CNV in the Ccl2(-/-) mice. CONCLUSIONS: These findings suggest that the lack of Ccl2 leads to a monocyte/macrophage-trafficking defect during aging and to an impaired recruitment of these cells to sites of laser injury. Other, previously described features of Ccl2(-/-) mice that are similar to AMD may be the result of aging alone.
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Envejecimiento/fisiología , Quimiocina CCL2/fisiología , Lipofuscina/metabolismo , Macrófagos/metabolismo , Degeneración Macular/metabolismo , Drusas Retinianas/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Factor de Crecimiento Epidérmico/metabolismo , Angiografía con Fluoresceína , Técnica del Anticuerpo Fluorescente Indirecta , Técnicas para Inmunoenzimas , Degeneración Macular/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oftalmoscopía , Drusas Retinianas/patologíaRESUMEN
BACKGROUND: Adeno-associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long-term therapy is the development of immune responses against the vector or transgene product. METHODS: We evaluated cellular and humoral responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression. RESULTS: Following subretinal administration of vector, splenocytes and T-cells from draining lymph nodes showed minimal activation following stimulation by co-culture with AAV2. Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose. NAbs were present in the serum of a proportion of mice receiving a higher dose of the vector. Furthermore, no differences in immunoglobulin titre in serum or ocular fluids against RPE65 protein or AAV2 capsid between treated and control mice were detected. Histological examination showed no evidence of retinal toxicity or leukocyte infiltration compared to uninjected eyes. Repeat administration of low-dose AAV.hRPE65.hRPE65 to both eyes of RPE65(-/-) mice resulted in transgene expression and functional rescue, but re-administration of high-dose AAV2 resulted in boosted NAb titres and variable transgene expression in the second injected eye. CONCLUSIONS: These data, which were obtained in mice, suggest that, following subretinal injection, immune responses to AAV2 are dose-dependent. Low-dose AAV2 is well tolerated in the eye, with minimal immune responses, and transgene expression after repeat administration of vector is achievable. Higher doses lead to the expression of NAbs that reduce the efficacy of repeated vector administration.